Method for producing and separating midbrain dopaminergic neurons

Abstract

The present disclosure provides methods of producing midbrain dopaminergic neurons (mDAs) and precursors thereof, mDAs and precursors thereof produced thereby, as well as compositions comprising these cells, and their use for the prevention and / or treatment of neurological diseases. The present disclosure also provides methods of isolating mDA and precursors thereof from cell populations using novel surface markers.

Description

[0001] (This application is a divisional application of application 202080075663.3, filed on August 31, 2020, entitled “Method for generating and isolating midbrain dopamine neurons”).

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 893674, filed August 29, 2019, the contents of which are incorporated herein by reference in their entirety, and claims priority thereto. Technical Field

[0004] This invention provides a method for generating midbrain dopamine neurons (mDA) and their precursors, the mDA and its precursors generated by this method, and compositions comprising such cells. This invention also provides the use of mDA and compositions thereof for the prevention and / or treatment of neurological diseases. This invention further provides a method for isolating mDA and its precursors from a cell population using novel surface markers. Background Technology

[0005] Previously, embryonic and somatic stem cells were 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, there have been research and technological advances 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.

[0006] Therefore, there is still a need for improved methods to generate midbrain dopamine neurons suitable for the treatment of neurodegenerative diseases such as Parkinson's disease. Summary of the Invention

[0007] This invention provides a method for generating midbrain dopamine neurons (mDA) and their precursors, mDA and their precursors generated by this method, compositions comprising such cells, and the use of such cells and compositions for the prevention and / or treatment of neurological diseases. Furthermore, this invention provides a method for isolating mDA and its precursors from a cell population using novel surface markers.

[0008] In some embodiments, the present invention provides a method for inducing stem cell differentiation in vitro, comprising: contacting stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of sound 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 differentiated cell population expressing at least one marker indicating a midbrain dopamine neuron (mDA) or its precursor, wherein the at least one activator of FGF signaling is selected from FGF18, FGF17, FGF8a, and combinations thereof.

[0009] In some embodiments, the present invention provides a method for inducing stem cell differentiation in vitro, comprising: contacting stem cells with at least one inhibitor of SMAD (Small Mothers Against Decapentaplegic) signaling, at least one activator of sound hedgehog factor (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 differentiated cell population expressing at least one marker indicating a midbrain dopamine neuron (mDA) or its precursor, wherein 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.

[0010] In some embodiments, the cells are contacted with at least one activator of FGF signaling for at least about 1 day. In some embodiments, the cells are contacted with at least one activator of FGF signaling for up to about 15 days. In some embodiments, the cells are contacted with at least one activator of FGF signaling for about 5 days.

[0011] In some embodiments, the initial contact of the cells with the at least one activator of FGF signaling is at least about 5 days after the initial contact of the cells with the at least one inhibitor of SMAD signaling.

[0012] In some embodiments, the initial contact of the cells with the at least one activator of FGF signaling is approximately 10 days after the initial contact of the cells with the at least one inhibitor of SMAD signaling. In some embodiments, the initial contact of the cells with the at least one activator of FGF signaling is 12 days after the initial contact of the cells with the at least one inhibitor of SMAD signaling.

[0013] In some embodiments, the cells are exposed to at least one inhibitor of SMAD signaling for approximately 5 days. In some embodiments, the cells are exposed to at least one inhibitor of SMAD signaling for 7 days.

[0014] In some embodiments, the cells are contacted with at least one activator of the SHH signaling pathway for approximately 5 days. In some embodiments, the cells are contacted with at least one activator of the SHH signaling pathway for 7 days.

[0015] In some embodiments, the cells are contacted with at least one activator of Wnt signaling for approximately 10 days. In some embodiments, the cells are contacted with at least one activator of Wnt signaling for 12 days.

[0016] In some embodiments, the concentration of the at least one Wnt signaling activator is increased approximately 4 days after initial contact with the stem cells. In some embodiments, the concentration of the at least one Wnt signaling activator is increased from approximately 300% to approximately 1000% compared to its initial concentration. In some embodiments, the concentration of the at least one Wnt signaling activator is increased to approximately 3 μM to 10 μM. In some embodiments, the concentration of the at least one Wnt signaling activator is increased to approximately 3 μM. In some embodiments, the concentration of the at least one Wnt signaling activator is increased to approximately 7.5 μM.

[0017] In some embodiments, the at least one activator for FGF signaling includes FGF18.

[0018] In some embodiments, the at least one inhibitor of SMAD signaling is selected from inhibitors of TGFβ / Activin-Nodal signaling, inhibitors of bone morphogenetic protein (BMP) signaling, and combinations thereof.

[0019] In some embodiments, the at least one TGFβ / Activin-Nodal signaling inhibitor comprises an ALK5 inhibitor.

[0020] In some embodiments, the at least one inhibitor of TGFβ / Activin-Nodal signaling comprises SB431542, or a derivative thereof, or a mixture thereof. In some embodiments, the derivative of SB431542 is A83-01. In some embodiments, the at least one inhibitor of TGFβ / Activin-Nodal signaling comprises SB431542.

[0021] In some embodiments, the at least one inhibitor of BMP signaling comprises LDN193189, Noggin, dorsomorphin, its derivatives, or mixtures thereof. In some embodiments, the at least one inhibitor of BMP comprises LDN-193189.

[0022] In some embodiments, the at least one Wnt signaling activator comprises an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling.

[0023] In some embodiments, the at least one Wnt signal activator is selected from CHIR99021, BIO, CHIR98014, lithium, 3F8, Wnt3A, Wnt1, Wnt5a, their derivatives, and mixtures thereof. In some embodiments, the at least one Wnt signal activator comprises CHIR99021.

[0024] In some embodiments, the activator of the at least one SHH signal is selected from SHH proteins, smoothing agonists (SAGs), derivatives thereof, and mixtures thereof. In some embodiments, the SHH protein comprises recombinant SHH, purified SHH, or a combination thereof.

[0025] In some embodiments, the recombinant SHH comprises a recombinant protein having at least about 80% identity with the N-terminal fragment of mouse sound hedgehog factor. In some embodiments, the recombinant SHH comprises SHH C25Ⅱ. In some embodiments, the SAG comprises purmorphamine.

[0026] In some embodiments, the at least one marker indicating a midbrain dopamine neuron or its precursor is selected from EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LMO3, SNCA, ADCAP1, CHRNA4, GIRK2, and combinations thereof.

[0027] In some embodiments, approximately 10 days after the stem cells are initially contacted with the at least one inhibitor of SMAD signaling, the differentiated cells have detectable levels of expression of at least one marker indicating midbrain dopamine neurons or their precursors.

[0028] In some embodiments, the differentiated cells exhibit detectable levels of EN1 expression approximately 30 days after the initial contact of the stem cells with at least one inhibitor of SMAD signaling. In some embodiments, the differentiated cells exhibit detectable levels of EN1 expression approximately 40 days after the initial contact of the stem cells with at least one inhibitor of SMAD signaling.

[0029] In some embodiments, the differentiated cells do not express at least one marker selected from the following: PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.

[0030] In some embodiments, the method further includes subjecting the differentiated cell population to conditions conducive to the differentiation of midbrain dopamine neuron precursors into midbrain dopamine neurons.

[0031] In some embodiments, the conditions include exposing the cells to at least one of the following: brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor β3 (TGFβ3), ascorbic acid (AA), and DAPT.

[0032] In some embodiments, the stem cells are selected from human, non-human primate, or rodent non-embryonic stem cells; human, non-human primate, or rodent embryonic stem cells; human, non-human primate, or rodent induced pluripotent stem cells; and human, non-human primate, or rodent recombinant pluripotent stem cells. In some embodiments, the stem cells are human stem cells. In some embodiments, the stem cells are pluripotent or multipotent stem cells. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are selected from embryonic stem cells, induced pluripotent stem cells, and combinations thereof.

[0033] In another aspect, the present invention provides a population of in vitro differentiated cells, wherein the in vitro differentiated cells are obtained by any of the foregoing methods.

[0034] In another aspect, the present invention provides a population of in vitro differentiated cells, wherein at least about 50% of the cells express at least one marker indicating midbrain dopamine neurons or their precursors, and less than about 50% of the differentiated cells express at least one marker selected from the following: PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof. In some embodiments, the at least one marker indicating midbrain dopamine neurons or their precursors is selected from EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2.

[0035] In another aspect, the present invention provides a composition comprising the cell population disclosed herein. In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0036] In another aspect, the present invention provides a method for isolating midbrain dopamine neurons and their precursors from a cell population, comprising isolating cells that do not express at least one negative surface marker at a detectable level but express at least one positive surface marker at a detectable level.

[0037] In another aspect, the present invention provides a method for isolating midbrain dopamine neurons and their precursors from a cell population, comprising isolating cells that: (a) do not express at least one negative surface marker at a detectable level 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 surface marker in the cell population.

[0038] In some embodiments, the at least one positive surface marker is selected from CD171, CD184, and combinations thereof. In some embodiments, the at least one positive surface marker comprises CD184. In some embodiments, the at least one negative surface marker is selected from CD49e, CD99, CD340, and combinations thereof. In some embodiments, the at least one negative surface marker comprises CD49e. In some embodiments, the method includes isolating cells that do not express detectable levels of CD49e but express detectable levels of CD184.

[0039] In some embodiments, the method includes isolating cells that do not express detectable levels of CD49e or express CD49e at a reduced level compared to the average expression level of CD49e in the cell population; and cells that express CD184 at an increased level compared to the average expression level of CD184 in the cell population.

[0040] In another aspect, the present invention provides a population of in vitro differentiated cells, wherein at least about 50% of the cells express at least one positive surface marker at a detectable level, but do not express at least one negative surface marker at a detectable level.

[0041] 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 at 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.

[0042] In some embodiments, the at least one positive surface marker is selected from CD171, CD184, and combinations thereof. In some embodiments, the at least one positive surface marker comprises CD184. In some embodiments, the at least one negative surface marker is selected from CD49e, CD99, CD340, and combinations thereof. In some embodiments, the at least one negative surface marker comprises CD49e. In some embodiments, the at least one positive surface marker comprises CD184, and the at least one negative surface marker comprises CD49e.

[0043] In another aspect, the present invention provides compositions comprising the cell populations disclosed herein. In some embodiments, the compositions disclosed herein are pharmaceutical compositions further comprising a pharmaceutically acceptable carrier. In another aspect, the present invention provides a kit for inducing differentiation of stem cells 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.

[0044] In some embodiments, the kit further includes (f) instructions for inducing stem cells to differentiate into a population of differentiated cells expressing at least one midbrain DA precursor marker.

[0045] In another aspect, the present invention provides a method for preventing and / or treating neurodegenerative diseases in a subject, comprising administering to the subject an effective amount of one of the following substances: (a) the cell population disclosed herein; or (b) the composition disclosed herein.

[0046] In some embodiments, the neurodegenerative disease is Parkinson's disease, Huntington's disease, Alzheimer's disease, or multiple sclerosis.

[0047] In some embodiments, the cell populations or compositions disclosed herein are used for the prevention and / or treatment of neurodegenerative diseases in a subject. In some embodiments, the neurodegenerative disease is Parkinson's disease, Huntington's disease, Alzheimer's disease, or multiple sclerosis. Attached Figure Description

[0048] Figure 1 This study demonstrates EN1 expression in stem cell-derived mDA and precursors from day 3 to day 30 using the Wnt-Boost protocol.

[0049] Figure 2 Describe a scheme that combines the use of FGF8b and Wnt-Boost.

[0050] Figure 3The EN1 protein was shown to be maintained in differentiated cells in a duration-dependent manner depending on FGF8b contact. Cells expressing EN1 also expressed FOXA2 and LMX1A.

[0051] Figures 4A-4B This shows the mRNA expression level of differentiated cells as measured by qRT-PCR on day 30. Figure 4A The mRNA expression level of EN1 was maintained in a duration-dependent manner with respect to FGF8b exposure, and the expression levels of FOXA2, NURR1, and TH were comparable under all conditions. Figure 4B This shows the mRNA expression levels of non-mDA markers (e.g., SMA and SIX1) induced in a duration-dependent manner by FGF8b contact.

[0052] Figure 5 The image shows SIX1 immunostaining of FGF8b-treated cells on day 30 of differentiation.

[0053] Figure 6 This shows the mRNA expression of markers in cells treated with FGFs on day 16 of differentiation.

[0054] Figure 7 Immunostaining of markers in cells treated with FGF8b and FGF18 on day 16 of differentiation.

[0055] Figure 8 RNA expression of markers was shown in cells treated with FGF8b and FGF18 at days 27 and 40 of differentiation.

[0056] Figure 9A This is a schematic diagram showing the donor vector structure of the NURR1::GFP reporter hPSC.

[0057] Figure 9B This shows the NURR1 mRNA level in cells differentiated from hPSCs using the Wnt-Boost protocol.

[0058] Figure 9C FACS results of midbrain DA neurons differentiated from H9-hPSC and NURR1::GFP hPSC on day 25 of differentiation.

[0059] Figure 10A Single-cell qRT PCR results shown in NURR1:GFP-positive cells isolated on day 25 and day 40 of differentiation.

[0060] Figure 10B Immunostaining of mDA sorted from NURR1 cells expressing TH, FOXA2, and NURR1 at day 60 of differentiation. Cells were sorted on day 25 and then cultured continuously until day 60.

[0061] Figure 11 Immunostaining images of NURR1::GFP-positive midbrain DA neurons transplanted in vivo 8 weeks after cell injection into immunodeficient mice are provided.

[0062] Figure 12 Immunostaining images of NURR1:GFP positive cells cultured according to the WNT-Boost protocol and the WNT-Boost+FGF18 protocol (days 12-16) are provided. Cells were sorted on day 25 and then cultured until day 40.

[0063] Figure 13A We present the expression of FOXA2, EN1, and NURR1 mRNA in NURR1:GFP-positive cells cultured according to the WNT-Boost protocol and the WNT-Boost+FGF18 (days 12-16) protocol. We compare the mRNA expression in sorted and unsorted cells.

[0064] Figure 13B Sorted NURR1:GFP-positive cells cultured according to the WNT-Boost protocol and the WNT-Boost+FGF18 (days 12-16) protocol were transplanted into immunodeficient mice. Neuronal process growth and TH and SC121 expression were detected in the ground cells.

[0065] Figure 14 The study showed that 390 surface markers were screened from mDA on day 25 of NURR1::GFP hPSC differentiation using the Wnt-Boost protocol. Antibodies were conjugated with PE, FITC, or APC.

[0066] Figure 15A The positive surface markers CD171 and CD184 were observed in NURRR1. + It accumulates in cells.

[0067] Figure 15B Provides RNA expression of CD171 and CD184 in differentiated cells using the Wnt-Boost protocol.

[0068] Figure 16A The negative surface markers CD49e, CD99, and CD340 were shown in NURRR1. + It accumulates in cells.

[0069] Figure 16B Provides RNA expression of CD49e, CD99, and CD340 in differentiated cells using the Wnt-Boost protocol.

[0070] Figure 17Cell morphologies were provided based on CD49e weakness or CD49e strength. Cells were sorted on day 25 of in vitro differentiation using the WNT-boost and WNT-boost+FGF18 protocols. After sorting, cells were cultured for another 15 days.

[0071] Figure 18 Immunostaining images of CD49e weak cells sorted on day 40 after in vitro differentiation according to the WNT-Boost and WNT-Boost+FGF18 protocols.

[0072] Figure 19 The results of CD49e-based FACS sorting of mDA derived from the hPSC cell line MEL1 are shown.

[0073] Figure 20 The morphology of MEL1-hPSC-derived mDA cells sorted according to CD49e weak or CD49e high values ​​is shown. Cells were sorted on day 25 of in vitro differentiation under the WNT-Boost and WNT-Boost+FGF18 protocols. After sorting, cells were cultured for another 15 days.

[0074] Figure 21 Immunostaining images of MEL1-hPSC-derived CD49e weak mDA on day 40 after in vitro differentiation under the WNT-Boost and WNT-Boost+FGF18 protocols. CD49e weak cells were sorted on day 25.

[0075] Figure 22 The relative mRNA expression in MEL1-hPSC-derived CD49e weak mDA was shown on day 40 after in vitro differentiation under the WNT-boost and WNT-boost+FGF18 protocols. CD49e weak cells were sorted on day 25.

[0076] Figure 23 The morphology of CD49e, CD99, or CD340 sorted cells on day 40 of in vitro differentiation under the WNT Boost+FGF18 protocol is shown. Cells were sorted on day 25 of in vitro differentiation.

[0077] Figure 24 The relative mRNA expression of CD49e, CD99, or CD340 in cells sorted on day 40 of in vitro differentiation under the WNT Boost+FGF18 protocol is shown. Cells were sorted on day 25 of in vitro differentiation.

[0078] Figure 25 The FACS sorting results of NURR1+ cells sorted according to 49E (PE) and 171 (APC) on day 25 of in vitro differentiation under the WNT-Boost protocol are shown.

[0079] Figure 26 The FACS sorting results of NURR1+ cells sorted according to 49E (PE) and 184 (APC) on day 25 of in vitro differentiation under the WNT-Boost protocol are shown.

[0080] Figure 27 Cell morphology is provided based on CD49e, CD171, and CD188. Cells were sorted on day 25 of in vitro differentiation using the WNT-Boost protocol. After sorting, cells were cultured for another 2 days.

[0081] Figure 28 The mRNA expression of cells sorted according to CD49e, CD171, and CD188 is shown. Cells were sorted on day 25 of in vitro differentiation using the WNT-Boost protocol. After sorting, cells were cultured for another 2 days.

[0082] Figure 29 The enrichment of the NURR1::GFP population in single CD49e weakly sorted cells was observed on day 25 of in vitro differentiation.

[0083] Figure 30 The enrichment of the NURR1:GFP population in CD49e weak CD184 high double-sorted cells was observed on day 25 of in vitro differentiation.

[0084] Figure 31 Display TH + Midbrain DA neurons co-express FOXA2 and GFP, suggesting that in Figure 30 Identity of midbrain DA neurons in a NURRR1:GFP population enriched in the medium.

[0085] Figure 32 The expression of midbrain DA marker mRNA in cells sorted by CD49e and CD184 was shown on day 25 of in vitro differentiation. After sorting, the cells were cultured in vitro for another 15 days.

[0086] Figure 33 The expression of non-midbrain DA mRNA in cells sorted by CD49e and CD184 was shown on day 25 of in vitro differentiation. After sorting, the cells were cultured in vitro for another 10 days.

[0087] Figure 34A-3 4D shows the in vivo survival rate of transplanted cells sorted using the CD markers (CD49e deletion and CD184 abundance) after in vitro differentiation under the WNT-Boost protocol. Figure 34A The results showed robust survival and TH levels of sorted cells in the graft compared to unsorted cells. + Cell enrichment. Figure 34B SOX2 was observed one month after transplantation.+ Precursor and KI67 + The number of dividing cells is reduced. Figure 34C shows... Figure 34B SOX2 + Quantification of stained cells. Figure 34D shows... Figure 34B Ki67 + Quantitative analysis of cells.

[0088] Figures 35A-35B The in vivo survival and EN1 expression of cells differentiated under the WNT-boost and WNT-boost+FGF18 regimens are shown. Figure 35A Showing the percentage of cells expressing EN1. Figure 35B The presence of striatal nerve innervation fibers at the transplantation site is shown. Detailed Implementation

[0089] This invention provides methods for generating mDA and its precursors, mDA and its precursors generated by such methods, compositions comprising such cells, and their use for the prevention and / or treatment of neurological diseases. Furthermore, this invention provides methods for isolating mDA and its precursors from cell populations using novel surface markers.

[0090] The present invention is based at least on the following finding: stem cell-derived mDA produced by the method of the present invention has sustained expression of EN1, for example, EN1 expression is maintained throughout the development and maturation of the mDA.

[0091] This specification and examples describe non-limiting implementations of the subject matter of this disclosure.

[0092] To clearly disclose rather than restrict, the detailed explanation is divided into the following sections:

[0093] 5.1. Definition;

[0094] 5.2. Stem cell differentiation methods;

[0095] 5.3. Methods for isolating midbrain dopamine neurons and their precursors;

[0096] 5.4. A composition comprising midbrain DA neurons and their precursors;

[0097] 5.5. Treatment methods for neurodegenerative diseases; and

[0098] 5.6. Reagent Kit

[0099] 5.1. Definition

[0100] The terms used in this specification generally have their ordinary meanings within the art, in the context of this disclosure, and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to practitioners in describing the compositions and methods of the invention and how they are made and used.

[0101] The term "about" or "approximately" refers to a range of acceptable error for a particular value as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may refer to within three or more standard deviations. Alternatively, "about" may refer to a range up to 20% of a given value, for example, up to 10%, up to 5%, or up to 1%. Or, particularly concerning biological systems or processes, the term may indicate within an order of magnitude, for example, within five times or two times a value.

[0102] As used herein, the term "signal transduction" in association with "signal transducing proteins" refers to proteins that are activated or otherwise influenced by ligands that bind to membrane receptor proteins or by other stimulatory factors. Examples of signal transducing proteins include, but are not limited to, SMAD, wingless (Wnt) complex proteins (including β-catenin), NOTCH, transforming growth factor β (TGFβ), Activin, Nodal, glycogen synthase kinase 3β (GSK3β) protein, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF). For many cell surface or internal receptor proteins, ligand-receptor interactions are not directly related to cellular responses. Before the ligand-activated receptor can produce the final physiological effect on cellular behavior, it must first interact with other intracellular proteins. Typically, the behavior of several interacting cellular protein chains changes after receptor activation or inhibition. The overall cellular changes resulting from receptor activation are called signal transduction mechanisms or signal transduction pathways.

[0103] As used in this article, the term "signal" refers to internal and external factors that control changes in cell structure and function. They can be chemical or physical in nature.

[0104] As used in this article, the term "ligand" refers to molecules and proteins that bind to receptors, such as transforming growth factor β (TFGP), Activin, Nodal, bone morphogenetic protein (BMP), etc.

[0105] As used herein, “inhibitor” refers to a compound or molecule (such as a small molecule, peptide, peptide-like substance, natural compound, siRNA, antisense nucleic acid, aptamer, or antibody) that interferes with (e.g., reduces, decreases, inhibits, eliminates, or blocks) the function of a molecular or pathway signaling. For example, an inhibitor can be any compound or molecule (e.g., including but not limited to the signaling molecules described herein) that alters the activity of a specified protein (signaling molecule, any molecule associated with the specified signaling molecule, or a specified associated molecule, such as glycogen synthase kinase 3β (GSK3β)), for example, by directly contacting SMAD signaling, contacting SMAD mRNA, causing a conformational change in SMAD, reducing SMAD protein levels, or interfering with the interaction of SMAD with signaling partners (e.g., including those described herein), and affecting the expression of SMAD target genes (e.g., those described herein).

[0106] Inhibitors also include molecules that indirectly regulate biological activity (e.g., SMAD activity) by intercepting upstream signaling molecules (e.g., within the extracellular domain; examples of signaling molecules and effectors include: blocking Noggin, a bone morphogenetic protein, inhibiting the activation of ALK receptors 1, 2, 3, and 6, thereby preventing downstream SMAD activation. Similarly, Chordin, Cerberus, and Follistatin are extracellular activators of SMAD signaling. Bambi, a transmembrane protein, can also act as a pseudoreceptor to block extracellular TGFβ signaling molecules). Antibodies that block upstream or downstream proteins can be considered to nullify extracellular activators of protein signaling. Inhibitors are described as competitive inhibition (binding to the active site by excluding or reducing the binding of another known binding compound) and allosteric inhibition (binding to a protein by interfering with the binding of a compound to the protein's active site, thereby altering the protein's conformation), as well as inhibition caused by binding to and affecting molecules upstream of a specified signaling molecule, thus leading to inhibition of the specified molecule. Inhibitors can also be "direct inhibitors" that inhibit a signaling target or signaling pathway by actually contacting the signaling target.

[0107] "Activator," as used herein, refers to a compound that increases, induces, stimulates, activates, promotes, or enhances the signal transduction function of molecules or pathways (e.g., Wnt signal transduction, SHH signal transduction, etc.).

[0108] As used herein, the term "WNT" or "wingless," when referring to ligand association, refers to a group of secreted proteins (i.e., integration 1 in humans) capable of interacting with WNT receptors, such as those in the Frizzled and LRPterailed / RYK receptor families. As used herein, the term "WNT or wingless signaling pathway" refers to a signaling pathway mediated by or not mediated by β-catenin, consisting of WnT family ligands and WnT family receptors (e.g., Frizzled and LRPterailed / RYK receptors). In some embodiments, the WNT signaling pathway includes mediated by β-catenin (e.g., WNT / -catenin).

[0109] As used in this article, the term "derivative" refers to a compound having a similar core structure.

[0110] As used herein, the term "population of cells" or "cell group" refers to a group consisting of at least two cells. In non-limiting examples, a cell group may 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, or at least about 1000 cells. The population may be a pure population containing only one cell type, such as a population of midbrain DA precursors or an undifferentiated stem cell population. Alternatively, the population may include more than one cell type, such as a mixed cell population.

[0111] As used in this article, the term "stem cell" refers to a cell that has the ability to divide indefinitely in culture and produce specialized cells.

[0112] As used herein, the terms “embryonic stem cell” and “ESC” refer to primitive (undifferentiated) cells derived from preimplantation embryos that are capable of dividing without differentiation for an extended period in culture and are known to develop into the three primary germ layers. Human embryonic stem cells refer to embryonic stem cells derived from human embryos. As used herein, the terms “human embryonic stem cell” or “hESC” refer to a pluripotent stem cell derived from early human embryos (including the blastocyst stage) that is capable of dividing without differentiation for an extended period in culture and developing into the three primary germ layers.

[0113] As used in this article, the term "embryonic stem cell line" refers to a population of embryonic stem cells cultured in vitro, which is allowed to proliferate without differentiation for days, months or years.

[0114] As used in this article, the term "totipotency" refers to the ability to produce all cell types in the body as well as all cell types that make up extraembryonic tissues (such as the placenta).

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

[0116] As used in this article, the term "multipotent" refers to the ability of an organism to develop into its three developmental germ layers (including the endoderm, mesoderm, and ectoderm).

[0117] As used herein, the term “induced pluripotent stem cell” or “iPSC” refers to a pluripotent stem cell formed by introducing certain embryonic genes (e.g., but not limited to OCT4, SOX2, and KLF4 transgenes) into somatic cells (see, for example, Takahashi and Yamanaka Cell 126, 663-676 (2006), which is incorporated herein by reference).

[0118] As used in this article, the term "somatic cell" refers to any cell in the body other than gametes (eggs or sperm); it is sometimes referred to as an "adult" cell.

[0119] As used in this article, the term "somatic (adult) stem cells" refers to relatively rare undifferentiated cells found in many organs and differentiated tissues, with limited self-renewal (laboratory) and differentiation capabilities.

[0120] As used herein, the term "neuron" refers to a nerve cell, the primary functional unit of the nervous system. A neuron consists of a cell body and its processes (an axon and at least one dendrite). Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

[0121] As used in this article, the term "proliferation" refers to an increase in the number of cells.

[0122] As used in this article, the term "undifferentiated" refers to cells that have not yet developed into a specific cell type.

[0123] As used herein, the term "differentiation" refers to the process by which undifferentiated embryonic cells acquire characteristics of specialized cells (such as neurons, heart, liver, or muscle cells). Differentiation is controlled by the interaction between cellular genes and extracellular physical and chemical conditions, typically through signaling pathways involving proteins embedded in the cell surface.

[0124] As used herein, the term “directed differentiation” refers to manipulating stem cell culture conditions to induce differentiation into a specific (e.g., desired) cell type, such as neural, neural crest, cranial plate, and non-neuroectodermal precursors. In stem cells, “directed differentiation” refers to using 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.

[0125] As used herein, the term “induced differentiation” in relation to cells refers to changing a default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Therefore, “induced stem cell differentiation” refers to inducing stem cells (such as human stem cells) to divide into daughter cells with characteristics different from those of stem cells, such as genotype (e.g., changes in gene expression determined by gene analysis (e.g., microarrays)) and / or phenotype (e.g., changes in the expression of protein markers of mDA or its precursors, such as EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2).

[0126] As used in this article, the term "cell culture" refers to the in vitro growth of cells in an artificial culture medium used for research or medical purposes.

[0127] As used herein, the term "culture medium" refers to a liquid that covers cells in a culture vessel, such as a Petri dish or a porous culture dish, and contains nutrients to nourish and support the cells. Growth factors may also be added to the culture medium to induce desired changes in the cells.

[0128] As used herein, the term “contact” with one or more cells using a compound (e.g., at least one inhibitor, activator, and / or inducer) means providing the compound at a location where the one or more cells are permitted to access the compound. Contact can be accomplished using any suitable method. For example, contact can be achieved by adding a concentrated form of the compound to cells or a population of cells (e.g., in a cell culture setting) to achieve the desired concentration. Contact can also be achieved by using the compound as a component of a formulation culture medium.

[0129] As used herein, the term "in vitro" refers to an artificial environment and the processes or reactions that occur in that environment. In vitro environments include, but are not limited to, test tubes and cell cultures.

[0130] As used in this article, the term "in vivo" refers to the natural environment (e.g., an animal or a cell) and the processes or reactions that occur in the natural environment, such as embryonic development, cell differentiation, neural tube formation, etc., as used in this article.

[0131] As used in this article, the term “expression” in relation to genes or proteins refers to the production of mRNA or proteins that can be observed using analyses such as microarray analysis, antibody staining analysis, etc.

[0132] As used herein, the term "marker" or "cell marker" refers to a gene or protein that identifies a specific cell or cell type. A cell's markers may not be limited to a single marker; a marker can refer to a "pattern" of markers that allows a specified set of markers to distinguish one cell or cell type from another.

[0133] As used herein, when referring to any cell disclosed herein, the terms “derived from,” “established from,” or “differentiated from” mean cells obtained from the final mother cell in a cell line (e.g., isolated, purified, etc.), tissues (e.g., isolated embryos), or any cells contained in cultured mother cells using any manipulating liquid, such as, but not limited to, single-cell isolation, in vitro culture, treatment (using, for example, proteins, chemicals, radiation, viral infection, DNA sequence transfection, such as using morphogenetics, etc.) and / or mutagenesis, or screening (e.g., by continuous culture). Derived cells can be selected from a mixed population by response to growth factors, cytokines, selective progression to cytokine treatment, adhesiveness, lack of adhesiveness, sorting processes, etc.

[0134] In this article, “individual” or “subject” refers to vertebrates, such as humans, or non-human animals, such as mammals. Mammals include, but are not limited to, humans, non-human primates, farm animals, livestock animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

[0135] As used in this article, the term "disease" refers to any symptom or condition that impairs or interferes with the normal function of cells, tissues, or organs.

[0136] As used herein, the terms “treatment” or “cure” refer to a clinical intervention that attempts to alter the course of a disease in the treated individual or cells, and may be used for prevention or in clinicopathological processes. Therapeutic effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, improvement or alleviation of disease status, and mitigation or improvement of prognosis. By preventing the progression of disease or symptoms, treatment can prevent deterioration of the condition in affected or diagnosed or suspected subjects, and treatment can also prevent the onset of the condition or symptoms in subjects at risk of the condition or suspected condition.

[0137] As used herein, the terms “negative,” “weak,” or “-” when used with respect to any surface marker disclosed herein mean that the surface marker (e.g., CD49e) is not expressed at a detectable level, or that its expression level in the cells is reduced compared to the average expression of the surface marker in the cell population from which cells were selected or sorted. As used herein, the terms “high,” “strong,” “+,” or “positive” when used with respect to any surface marker disclosed herein mean that the surface marker (e.g., CD184) is expressed at a detectable level or at an increased level compared to the average expression of the surface marker in the cell population.

[0138] In some embodiments, as known to those skilled in the art or of ordinary skill, cells are distinguished based on easily identifiable differences in staining intensity according to the expression level of cell surface markers. In some embodiments, a cutoff line for designating cells as “weak,” “negative,” or “-” cells for surface markers can be set based on the observed distribution of staining intensity (e.g., fluorescence intensity distribution) across all cells, wherein cells with staining intensities below about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% are designated as “weak,” “negative,” or “-” cells for surface markers. In some embodiments, a cutoff line for designating cells as “strong,” “high,” or “+” cells for surface markers can be set based on the observed distribution of staining intensity (e.g., fluorescence intensity distribution) across all cells, wherein cells with staining intensities above about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% are designated as “strong,” “high,” “+,” or “positive” cells for surface markers. In some implementations, the frequency distribution of staining for surface markers of all cells is obtained, and population curves are adapted to populations with higher and lower staining, and cells are assigned to the population to which they are statistically most likely to belong, based on a statistical analysis of the distribution of their respective populations.

[0139] 5.2. Stem cell differentiation methods

[0140] The present invention provides a method for inducing stem cell differentiation, comprising contacting stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling (referred to as "SMAD inhibitor"), at least one activator of sound hedgehog factor (SHH) signaling (referred to as "SHH activator"), and at least one activator of wingless (Wnt) signaling (referred to as "Wnt activator"); and further contacting the cells with at least one activator of fibroblast growth factor (FGF) signaling (referred to as "FGF activator") to obtain a cell population comprising differentiated cells expressing at least one marker indicating mDA or an mDA precursor.

[0141] In some embodiments, the at least one FGF activator can promote midbrain development. In some embodiments, the at least one FGF activator is selected from FGF8a, FGF17, FGF18, FGF2, and FGF4. In some embodiments, the at least one FGF activator is selected from FGF8a, FGF17, and FGF18. In some embodiments, the at least one FGF activator comprises FGF18.

[0142] In some embodiments, the initial contact of the cells with at least one activator of FGF signaling is at least about 5 days after the initial contact of the cells with at least one inhibitor of SMAD signaling. In some embodiments, the initial exposure of the cells to at least one FGF activator is at least about 10 days after the initial exposure of the stem cells to at least one SMAD inhibitor. In some embodiments, the FGF activator is selected from FGF8a, FGF17, FGF18, FGF8b, FGF2, and FGF4. Exposing cells to at least one FGF activator prolongs EN1 expression in differentiated cells.

[0143] In some embodiments, at least one marker indicating mDA or mDA precursor is selected from EN1, FOX1A, LMX1A, OTX2, NURR1, TH, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2.

[0144] In some embodiments, the concentration of the at least one Wnt activator is increased during its exposure to cells. In some embodiments, the concentration of the at least one Wnt activator begins to increase approximately 4 days after the stem cells are initially exposed to the at least one SMAD inhibitor. In some embodiments, the concentration of the at least one Wnt activator is increased by approximately 300% to approximately 1000%. In some embodiments, the cells are exposed to at least one Wnt activator with the increased concentration for at least approximately 7 days. In some embodiments, at least one additional Wnt activator is added to increase the total concentration of Wnt activators.

[0145] In some embodiments, the method further includes contacting the cells with midbrain DA lineage-specific activators and inhibitors (e.g., BDNF, GDNF, cAMP, TGFβ, ascorbic acid (AA), and / or DAPT) to induce mDA precursors to differentiate into mDA.

[0146] 5.2.1. Stem cells

[0147] This disclosure provides methods for inducing stem cell differentiation in vitro to generate mDA and its precursors. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are selected from embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), and combinations thereof. In some embodiments, the stem cells are multipotent stem cells. Non-limiting examples of stem cells that can be used with the methods of this disclosure include human, non-human primate, or rodent non-embryonic stem cells, embryonic stem cells, induced non-embryonic pluripotent cells, and engineered pluripotent cells. In some embodiments, the stem cells are human stem cells. Non-limiting examples of human stem cells include human embryonic stem cells (hESCs), human pluripotent stem cells (hPSCs), human induced pluripotent stem cells (hiPSCs), human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, ectodermal stem cells, F-class pluripotent stem cells, somatic stem cells, cancer stem cells, or any other cells capable of lineage-specific differentiation. In some embodiments, the stem cells are human embryonic stem cells (hESCs). In some embodiments, the stem cells are human induced pluripotent stem cells (hiPSCs).

[0148] In some embodiments, the stem cells or their progeny cells contain introduced heterologous nucleic acids, wherein the nucleic acids may encode desired nucleic acid or protein products or have informational value (e.g., see U.S. Patent No. 6,312,911, which is incorporated herein by reference in its entirety). Non-limiting examples of protein products include biomarkers detectable by in vivo imaging studies, such as receptors or other cell membrane proteins. Non-limiting examples of biomarkers 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)), β-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), enzymes (such as oxidases and peroxidases), and antigen molecules. As used herein, the term "reporter gene" or "reporter construct" refers to a genetic construct containing nucleic acid encoding a protein that is easily detected or easily analyzed, such as a colored protein, a fluorescent protein (e.g., GFP), or an enzyme (e.g., β-galactosidase (lacZ gene)). In some embodiments, the reporter may be driven by a recombinant promoter of a marker gene for precocious mitosis midbrain DA neurons (e.g., NURR1).

[0149] 5.2.2. SMAD Inhibitors

[0150] Non-limiting examples of SMAD inhibitors include inhibitors of transforming growth factor β (TGFβ) / Activin-Nodal signaling (referred to as "TGFβ / Activin-Nodal inhibitors") and inhibitors of bone morphogenetic protein (BMP) signaling. In some embodiments, the TGFβ / Activin-Nodal inhibitors may inactivate ligands (including TGFβ, BMP, Nodal, and Activin) and / or block their signaling pathways by blocking receptors and downstream effectors. Non-limiting examples of TGFβ / 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 some embodiments, the at least one TGFβ / Activin-Nodal inhibitor is selected from ALK5 inhibitors, ALK4 inhibitors, ALK7 inhibitors, and combinations thereof. In some embodiments, the TGFβ / Activin-Nodal inhibitor comprises an ALK5 inhibitor. In some embodiments, the TGFβ / Activin-Nodal inhibitor is a small molecule selected from SB431542, its derivatives, and mixtures thereof. "SB431542" refers to CAS number 301836-41-9, with the molecular formula C5. 22 H 18 N4O3, and molecules named 4-[4-(1,3-benzodioxolane-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide, for example, see the following structure:

[0151]

[0152] In some embodiments, the TGFβ / Activin-Nodal inhibitor comprises SB431542. In some embodiments, the TGFβ / Activin-Nodal inhibitor comprises a derivative of SB431542. In some embodiments, the derivative of SB431542 is A83-01.

[0153] In some 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 l; 30(7):715-20, all of which are incorporated herein by reference in their entirety. In some embodiments, the BMP inhibitor is a small molecule selected from LDN193189, Noggin, doxorphine, derivatives thereof, and mixtures thereof. "LDN193189" refers to the small molecule DM-3189, with the IUPAC name 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline and the chemical formula C. 25 H 22 N6, as shown in the following formula.

[0154]

[0155] LDN193189 acts as an inhibitor of SMAD signaling. It is also a highly potent small-molecule inhibitor of ALK2, ALK3, and ALK6 protein tyrosine kinases (PTKs), inhibiting the signaling of type I TGFβ receptors ALK1 and ALK3 family members, thereby suppressing the transmission of multiple biological signals, including bone morphogenetic protein (BMP) signals (BMP2, BMP4, BMP6, BMP7 and Activin cytokine), and subsequently SMAD phosphorylation of Smad1, Smad5, and Smad8 (Yu et al. (2008) Nat Med 14:1363-1369; Cuny et al. (2008) Bioorg. Med. Chem. Lett. 18:4388-4392, incorporated herein by reference).

[0156] In some embodiments, the BMP inhibitor comprises LDN193189. In some embodiments, the BMP inhibitor comprises Noggin.

[0157] In some embodiments, the stem cells are exposed to a SMAD inhibitor, such as a TGFβ / Activin inhibitor. In some embodiments, the TGFβ / Activin-Nodal inhibitor is SB431542 or A83-01. In some embodiments, the stem cells are exposed to two SMAD inhibitors. In some embodiments, the two SMAD inhibitors are a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor. In some embodiments, the stem cells are exposed to SB431542 or A83-01, and LDN193189 or Noggin. In some embodiments, the stem cells are exposed to both SB431542 and LDN193189.

[0158] In some embodiments, the stem cells are exposed to at least one SMAD inhibitor for at least about 5 days, or at least about 10 days. In some embodiments, the stem cells are exposed to at least one SMAD inhibitor for up to about 5 days or up to about 10 days. In some embodiments, the stem cells are exposed to at least one SMAD inhibitor for about 5 days to about 10 days. In some embodiments, the stem cells are exposed to at least one SMAD inhibitor for about 5 days. In some embodiments, the stem cells are exposed to at least one SMAD inhibitor for 7 days. In some embodiments, the cells are exposed to at least one SMAD inhibitor from day 0 to day 6. In some embodiments, at least one SMAD inhibitor is added to the cell culture medium containing the stem cells daily or every other day from day 0 to day 6. In some embodiments, at least one SMAD inhibitor is added to the cell culture medium containing the stem cells daily from day 0 to day 6.

[0159] In some embodiments, the cells are contacted or exposed to a TGFβ / Activin inhibitor. In some embodiments, the concentration of the TGFβ / Activin-Nodal inhibitor in contact with or exposure to the cells is between about 1 μM and about 20 μM, about 1 μM and about 10 μM, about 1 μM and about 15 μM, about 10 μM and about 15 μM, about 5 μM and about 10 μM, about 5 μM and about 15 μM, about 5 μM and about 20 μM, or about 15 μM and about 20 μM. In some embodiments, the concentration of the TGFβ / Activin-Nodal inhibitor in contact with or exposure to the cells is between about 1 μM and about 10 μM. In a particular embodiment, the concentration of the TGFβ / Activin-Nodal inhibitor in contact with or exposure to the cells is about 5 μM. In some embodiments, the concentration of the TGFβ / Activin-Nodal inhibitor in contact with or exposure to the cells is about 10 μM. In some embodiments, the TGFβ / Activin-Nodal inhibitor comprises SB431542 or a derivative thereof (e.g., A83-01). In some embodiments, the TGFβ / Activin-Nodal inhibitor comprises SB431542.

[0160] In some embodiments, the cells are contacted with or exposed to a BMP inhibitor. In some embodiments, the concentration of the BMP inhibitor in contact 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 some embodiments, the concentration of the BMP inhibitor in contact with or exposed to the cells is between about 200 nM and about 300 mM. In some embodiments, the concentration of the BMP inhibitor in contact with or exposure to cells is about 150 nM, about 200 nM, about 250 nM, about 300 nM, or about 350 nM. In some embodiments, the concentration of the BMP inhibitor in contact with or exposure to cells is about 250 nM. In some embodiments, the BMP inhibitor comprises LDN193189 or a derivative thereof. In some embodiments, the BMP inhibitor comprises LDN193189.

[0161] In some embodiments, cells are simultaneously exposed to or treated with a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor. In some embodiments, the stem cells are exposed to or treated with a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor for 7 days. In some embodiments, the cells are exposed to or treated with a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor from day 0 to day 6. In some embodiments, from day 0 to day 6, a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor are added to the cell culture medium containing the stem cells daily or every other day. In some embodiments, from day 0 to day 6, a TGFβ / Activin-Nodal inhibitor and a BMP inhibitor are added to the cell culture medium containing the stem cells daily.

[0162] 5.2.3. Wnt Activator

[0163] In some embodiments, the at least one Wnt activator reduces GSK3β to activate Wnt signaling. Therefore, in some embodiments, the Wnt activator is a GSK3β inhibitor. GSK3β inhibitors can activate the WNT signaling pathway; see, for example, Cadigan et al., J Cell Sci 2006; 119:395-402; Kikuchi et al., Cell Signaling. 2007; 19:659-671, which are incorporated herein by reference in their entirety. As used herein, the term “glycogen synthase kinase 3β inhibitor” or “GSK3β inhibitor” refers to a compound that inhibits the enzyme glycogen synthase kinase 3β; see, for example, Doble et al., J Cell Sci. 2003; 116:1175-1186, which are incorporated herein by reference in their entirety.

[0164] Non-limiting examples of Wnt activators or GSK3β inhibitors include CHIR99021, Wnt3A, Wnt1, Wnt5a, BIO((3E)-6-bromo-3-[3-(hydroxyamino)indol-2-ylidene]-1H-indol-2-one), CHIR98014, lithium, 3F8, and those disclosed in WO2011 / 149762, WO13 / 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 herein by reference in their entirety. In some embodiments, the at least one Wnt activator is a small molecule selected from CHIR99021, Wnt3A, Wnt1, Wnt5a, BIO, CHIR98014, lithium, 3F8, its derivatives, and mixtures thereof. In some embodiments, the at least one Wnt activator comprises CHIR99021 or a derivative thereof. In some embodiments, the at least one Wnt activator comprises CHIR99021. “CHIR99021” (also known as “aminopyrimidine” or “3-[3-(2-carboxyethyl)-4-methylpyrrole-2-methylene(methylidenyl)]-2-indolone”) refers to the IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile, with the following formula.

[0165]

[0166] CHIR99021 exhibits high selectivity, showing nearly a thousandfold selectivity for a group of related and unrelated kinases, with an IC50 of 6.7 nM for human GSK3β and nanomolar-level IC50 values ​​for rodent GSK3β homologues.

[0167] In some 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 some 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 some embodiments, the cells are contacted or exposed to at least one Wnt activator for about 5 to about 20 days, about 5 to about 15 days, about 10 to about 20 days, about 5 to about 15 days, or about 10 to about 15 days. In some embodiments, the cells are contacted with at least one Wnt activator for about 10 to about 15 days. In some embodiments, the cells are contacted with at least one Wnt activator for about 10 days. In some embodiments, the stem cells are contacted with at least one Wnt signaling activator for 12 days. In some embodiments, the cells are contacted with at least one Wnt activator from day 0 to day 11. In some embodiments, at least one Wnt activator is added to the cell culture medium containing the cells daily or every other day from day 0 to day 11.

[0168] In some embodiments, the concentration of the at least one Wnt activator is increased during cell exposure (also referred to as "Wnt Boost"). In some embodiments, Wnt Boost is increased for at least about 2 days, at least about 4 days, or at least about 5 days after initial cell exposure to the at least one Wnt activator. In some embodiments, Wnt Boost is increased for about 4 days after initial cell exposure to the at least one Wnt activator.

[0169] In some embodiments, the cells are contacted or exposed to an increased concentration of at least one Wnt activator for at least about 5 days or at least about 10 days. In some embodiments, the cells are contacted or exposed to an increased concentration of at least one Wnt activator for at least about 5 days. In some embodiments, the cells are contacted with an increased concentration of at least one Wnt activator for up to about 5 days, up to about 10 days, or up to about 15 days. In some embodiments, the cells are contacted with an increased concentration of at least one Wnt activator for up to about 10 days.

[0170] In some embodiments, the cells are exposed to an increased concentration of at least one Wnt activator for about 5 to 15 days, or about 5 to 10 days, or about 10 to 15 days. In some embodiments, the cells are exposed to an increased concentration of at least one Wnt activator for about 5 to 10 days. In some embodiments, the cells are exposed to an increased concentration of at least one Wnt activator for about 5 days, about 10 days, or about 15 days. In some embodiments, the cells are exposed to an increased concentration of at least one Wnt activator for about 5 days. In some embodiments, the cells are exposed to an increased concentration of at least one Wnt activator for 6 days. In some embodiments, the cells are exposed to an increased concentration of at least one Wnt activator from day 4 to day 9. In some embodiments, the cells are exposed to an increased concentration of at least one Wnt activator for about 10 days. In some embodiments, the cells are exposed to an increased concentration of at least one Wnt activator for 8 days. In some implementations, from day 4 to day 11, the cells are contacted or exposed to an increased concentration of at least one Wnt activator.

[0171] In some embodiments, the initial concentration of at least one Wnt activator that contacts or exposes the cells prior to Wnt Boost is less than about 5 μM, less than about 3 μM, or less than about 1 μM, including but not limited to between about 0.01 μM and about 5 μM, between about 0.01 μM and about 3 μM, between about 0.05 μM and about 3 μM, between about 0.1 μM and about 3 μM, between about 0.5 μM and about 3 μM, between about 0.5 μM and about 2 μM, or between about 0.5 μM and about 1 μM. In some embodiments, the initial concentration of at least one Wnt activator that contacts or exposes the cells prior to Wnt Boost is less than about 1 μM, for example, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, or about 1 μM. In some embodiments, the initial concentration of at least one Wnt activator that is in contact with or exposed to cells before Wnt Boost is approximately 0.5 μM. In some embodiments, the initial concentration of at least one Wnt activator that is in contact with or exposed to cells before Wnt Boost is approximately 0.7 μM.

[0172] In some embodiments, the increased concentration of the at least one Wnt activator after Wnt Boost is about 3 μM or higher, about 5 μM or higher, about 10 μM or higher, about 15 μM or higher, or about 20 μM or higher. In some embodiments, the increased concentration of the at least one Wnt activator after Wnt Boost is between about 3 μM and about 15 μM, between about 3 μM and about 10 μM, or between about 5 μM and about 10 μM. In some embodiments, the increased concentration of the at least one Wnt activator after Wnt Boost is about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, or about 10 μM. In some embodiments, the increased concentration of the at least one Wnt activator after Wnt Boost is approximately 3 μM. In some embodiments, the increased concentration of the at least one Wnt activator after Wnt Boost is approximately 7 μM. In some embodiments, the increased concentration of the at least one Wnt activator after Wnt Boost is approximately 7.5 μM.

[0173] In some embodiments, the concentration of the at least one Wnt activator is increased by about 50% to about 2000%, or about 100% to about 1500%, or about 150% to about 1500%, or about 200% to about 1500%, or about 250% to about 1500%, or about 300% to about 1500%, or about 300% to about 1000%, or about 300% to about 400%, or about 500% to about 1000%, or about 800% to about 1000%, or about 900% to about 1000%, or about 950% to about 1000%. In some embodiments, the concentration of the at least one Wnt activator is increased by about 300% to about 1000% compared to the initial concentration of the Wnt activator in contact with or exposure to the cells. In some embodiments, the concentration of the at least one Wnt activator is increased by about 300% to about 400% compared to the initial concentration upon contact with or exposure to cells. In some embodiments, the concentration of the at least one Wnt activator is increased by about 900% to about 1000% compared to the initial concentration upon contact with or exposure to cells. In some embodiments, the concentration of the at least one Wnt activator is increased by about 300%, about 350%, about 400%, about 450%, about 500%, about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, about 1000%, about 1050%, or about 1100% compared to the initial concentration upon contact with or exposure to cells. In some embodiments, the concentration of the at least one Wnt activator is increased by about 300% compared to the initial concentration upon contact with or exposure to cells. In some embodiments, the concentration of the at least one Wnt activator is increased by about 350% compared to the initial concentration upon contact with or exposure to the cells. In some embodiments, the concentration of the at least one Wnt activator is increased by about 950% compared to the initial concentration upon contact with or exposure to the cells. In some embodiments, the concentration of the at least one Wnt activator is increased by about 1000% compared to the initial concentration upon contact with or exposure to the cells.

[0174] In some embodiments, the at least one Wnt activator comprises a GSK3β inhibitor. In some embodiments, the at least one Wnt activator comprises CHIR99021 or a derivative thereof. In some embodiments, the at least one Wnt activator comprises CHIR99021.

[0175] 5.2.4. SHH Activator

[0176] As used herein, the terms “hedgehog factor,” “SHH,” or “Shh” refer to a protein of one of at least three proteins in the mammalian signaling pathway family called hedgehog factors, another being the desert hedgehog factor (DHH), and the third being the Indian hedgehog factor (IHH). SHH interacts with at least two transmembrane proteins by interacting with the transmembrane molecules patched (PTC) and smoothed (SMO). SHH typically binds to PTC, which allows SMO to be activated as a signaling sensor. In the absence of SHH, PTC typically inhibits SMO, which in turn activates transcriptional repressors, thus preventing the transcription of certain genes. When SHH is present and bound to PTC, PTC cannot interfere with SMO function. In the absence of SMO inhibition, 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 some embodiments, the SHH activator refers to any molecule or compound capable of activating the SHH signaling pathway, including molecules or compounds capable of binding PTC or SMO. In some embodiments, the SHH activator is selected from molecules that bind PCT, molecules that bind SMO, and combinations thereof. Non-limiting examples of SHH activators include WO10 / 096496, WO13 / 067362, Chambers et al., Nat Biotechnol. 2009 Mar; 27(3):275-80, and Kriks et al., Nature. 2011 Nov 6; 480(7378):547-51. In some embodiments, the SHH activator comprises an SHH protein, an SMO agonist, or a combination thereof. In some embodiments, the SHH protein comprises recombinant SHH, purified SHH, or a combination thereof. In some embodiments, the recombinant SHH comprises SHH with mouse SHH. The N-terminal fragment has at least about 80%, about 85%, about 90%, about 95%, or about 99% identity with the recombinant protein. In some embodiments, the recombinant SHH comprises SHH C25Ⅱ. In some embodiments, the SMO agonist comprises puromorphamine.

[0177] In some 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 some 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 some embodiments, the cells are contacted or exposed to the at least one SHH activator for between about 5 days and about 10 days. In some embodiments, the cells are contacted or exposed to the at least one SHH activator for about 5 days. In some embodiments, the cells are contacted or exposed to the at least one SHH activator for 7 days. In some embodiments, the cells are contacted or exposed to the at least one SHH activator from day 0 to day 6. In some embodiments, at least one SHH activator is added to the cell culture medium containing the cells daily from day 0 to day 6. In some embodiments, at least one SHH activator is added to the cell culture medium containing the cells daily from day 0 to day 6.

[0178] In some embodiments, the concentration of the at least one SHH activator that contacts or exposes the cells is between about 50 ng / mL and about 1000 ng / mL, about 100 ng / mL and about 1000 ng / mL, about 20 ng / mL and about 1000 ng / mL, about 300 ng / mL and about 1000 ng / mL, about 400 ng / mL and about 1000 ng / mL, about 500 ng / mL and about 1000 ng / mL, about 400 ng / mL and about 800 ng / mL, about 400 ng / mL and about 700 ng / mL, about 400 ng / mL and about 600 ng / mL, or about 500 ng / mL and about 600 ng / mL. In some embodiments, the concentration of the at least one SHH activator that contacts or exposes the cells is between about 400 ng / mL and about 600 ng / mL. In some embodiments, the concentration of the at least one SHH activator that contacts or exposes the cells is about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 550 ng / mL, or about 600 ng / mL. In some embodiments, the concentration of the at least one SHH activator that contacts or exposes the cells is about 500 ng / mL.

[0179] In some embodiments, the at least one SHH signal transduction activator comprises SHH C25Ⅱ.

[0180] 5.2.5. FGF activator

[0181] The FGF family includes secretory signaling proteins (secretory FGFs) that signal receptor tyrosine kinases. Phylogenetic analysis shows that the 22 FGF genes can be divided into seven subfamilies, each containing two to four members. Branch length is proportional to the evolutionary distance between each gene.

[0182] In some embodiments, the FGF activator is selected from FGF8a, FGF17, FGF18, FGF8b, FGF2, FGF4, and their derivatives. In some embodiments, the FGF activator is selected from FGF8a, FGF17, FGF18, FGF2, FGF4, and their derivatives. In some embodiments, the FGF activator is selected from FGF8a, FGF17, and FGF18.

[0183] The FGF8 subfamily consists of FGF8a, FGF8b, FGF17, and FGF18. Early patterns of the midbrain and cerebellum in vertebrates are regulated by midbrain / hindbrain organizers that produce FGF8a, FGF8b, FGF17, and FGF18. Studies have shown that FGF8b functions differently from FGF8a, FGF17, and FGF18 (Liu et al., Development. 2003 Dec; 130(25):6175-85). FGF8b is the only protein capable of inducing the rl gene Gbx2 and strongly activating the pathway inhibitor Spry 1 / 2, as well as inhibiting the midbrain gene Otx2 (Liu 2003). Furthermore, FGF8b extends organizers along the connections between the induced Gbx2 domain and the remaining Otx2 region in the midbrain, and is associated with cerebellar development (Liu 2003). In contrast, FGF8a, FGF17, and FGF18 lead to midbrain expansion and upregulation of midbrain gene expression (Liu 2003).

[0184] In some embodiments, the FGF activator can induce midbrain expansion and upregulate midbrain gene expression. In some embodiments, the FGF activator is selected from FGF8a, FGF17, FGF18, FGF2, FGF4, their derivatives, and combinations thereof. In some embodiments, the FGF activator comprises or is FGF18.

[0185] In some embodiments, the cells are exposed to or contacted with 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 some embodiments, the cells are exposed to or contacted with the at least one FGF activator for up to about 5 days, up to about 10 days, up to about 15 days, or up to about 20 days. In some embodiments, the cells are exposed to or contacted with the at least one FGF activator for about 1 day to about 20 days, about 1 day to about 15 days, about 5 days to about 20 days, about 5 days to about 15 days, about 5 days to about 10 days, or about 10 days to about 20 days. In some embodiments, the cells are exposed to or contacted with the at least one FGF activator for about 5 days to about 10 days. In some embodiments, the cells are exposed to or contacted with the at least one FGF activator for about 3 days, about 5 days, or about 8 days. In some embodiments, the cells are exposed to or contacted with the at least one FGF activator for about 5 days.

[0186] In some embodiments, the initial contact or initial exposure of the cells with the at least one FGF activator is at least about 5 days or at least about 10 days after the initial contact or initial exposure of the cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of the cells with 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 after the initial contact or initial exposure of the cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of the cells with 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 after the initial contact or initial exposure of the cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of the cells with the at least one FGF activator is between about 5 days and about 10 days after the initial contact or initial exposure of the cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of the cells with the at least one FGF activator is approximately 10 days from the initial contact or initial exposure of the cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of the cells with the at least one FGF activator is approximately 9 days from the initial contact or initial exposure of the cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of the cells with the at least one FGF activator is 10 days from the initial contact or initial exposure of the cells with the at least one SMAD inhibitor. In some embodiments, the initial contact or initial exposure of the cells with the at least one FGF activator is 12 days from the initial contact or initial exposure of the cells with the at least one SMAD inhibitor.

[0187] In some embodiments, the initial contact or initial exposure of the cells with the at least one FGF activator is approximately 5 days from the initial contact or initial exposure of the cells with the at least one SMAD inhibitor, and the cells are exposed to the at least one FGF activator for approximately 3 days. In some embodiments, the initial contact or initial exposure of the cells with the at least one FGF activator is approximately 5 days from the initial contact or initial exposure of the cells with the at least one SMAD inhibitor, and the cells are exposed to the at least one FGF activator for approximately 5 days. In some embodiments, the initial contact or initial exposure of the cells with the at least one FGF activator is approximately 10 days from the initial contact or initial exposure of the cells with the at least one SMAD inhibitor, and the cells are exposed to the at least one FGF activator for approximately 3 days. In some embodiments, the initial contact or initial exposure of the cells with the at least one FGF activator is approximately 10 days from the initial contact or initial exposure of the cells with the at least one SMAD inhibitor, and the cells are exposed to the at least one FGF activator for approximately 5 days. In some embodiments, the initial contact or initial exposure of the cells with the at least one FGF activator is 12 days from the initial contact or initial exposure of the cells with the at least one SMAD inhibitor, and the cells are exposed to the at least one FGF activator for 5 days.

[0188] In some embodiments, the concentration of the at least one FGF activator that contacts or exposes the cells is between about 10 ng / mL and about 500 ng / mL, between about 50 ng / mL and about 500 ng / mL, between about 100 ng / mL and about 500 ng / mL, between about 100 ng / mL and about 400 ng / mL, between about 100 ng / mL and about 300 ng / mL, between about 100 ng / mL and about 200 ng / mL, or between about 100 ng / mL and about 250 ng / mL. In some embodiments, the concentration of the at least one FGF activator that contacts or exposes the cells is between about 100 ng / mL and about 200 ng / mL, and the concentration of the at least one FGF activator that contacts or exposes the cells is about 100 ng / mL. In some embodiments, the concentration of the at least one FGF activator that contacts or exposes the cells is about 200 ng / mL.

[0189] In some embodiments, the at least one FGF activator comprises FGF18.

[0190] In some non-limiting embodiments, the stem cells are contacted or exposed to at least one TGFβ / Activin-Nodal inhibitor (e.g., SB431542, e.g., at a concentration of about 10 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 C25Ⅱ, e.g., at a concentration of about 500 ng / mL) for about 5 days (e.g., 7 days, e.g., from day 0 to day 6), and the cells are contacted with the at least one Wnt activator (e.g., CHIR99021, e.g., at a concentration of about 0.7 μM for 5 days (e.g., 4 days, e.g., from day 0 to day 3), and at a concentration of about 7.5 μM for about 5 days (e.g., 6 days, e.g., from day 4 to day 9), and at a concentration of about 3 μM for about 2 days (e.g., from day 10 to day 11)). The cells are contacted 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 from about 10 days (e.g., 10 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).

[0191] 5.2.6. Cell Culture Medium

[0192] In some embodiments, the above-described inhibitors and activators are added to the cell culture medium containing the cells. Suitable cell culture media include, but are not limited to, those described above. Serum Replacement (“KSR”) medium, Culture media (NB), N2 medium, B-27 medium and (“E8 / E6”) media, and combinations thereof. KSR medium, NB medium, N2 medium, B-27 medium, and E8 / E6 medium are all commercially available. KSR medium is a defined serum-free formulation optimized for the growth and maintenance of undifferentiated hESCs in a medium.

[0193] In some embodiments, the cell culture medium is KSR medium. The composition of KSR medium is disclosed in WO2011 / 149762. In some embodiments, the KSR medium comprises Knockout DMEM, Knockout Serum Replacement, L-glutamine, Pen / Strep, MEM, and 13-mercaptoethanol. In some 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 μM 13-mercaptoethanol.

[0194] In some 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. Furthermore, E8 / E6 medium can serve as a basis for custom culture medium formulations for PSC culture. An example E8 / E6 medium 9 is described in Chen et al., Nat Methods 2011 May; 8(5):424-9, which is incorporated herein by reference in its entirety. An exemplary E8 / E6 medium is disclosed in WO 15 / 077648, which is incorporated herein by reference in its entirety. In some embodiments, the E8 / E6 cell culture medium contains DMEM / F12, ascorbic acid, selenium, insulin, NaHCO3, transferrin, FGF2, and TGFβ. The difference between E8 / E6 medium and KSR medium is that E8 / E6 medium does not contain active BMP or Wnt components. Therefore, in some embodiments, when the stem cell populations of this disclosure are cultured in E8 / E6 medium to differentiate into proprioceptor populations, it is not necessary to add at least one inhibitor of SMAD signaling (e.g., an inhibitor of BMP) to the E8 / E6 medium.

[0195] 5.2.7. Differentiated Cells

[0196] In some embodiments, the method includes obtaining a population of differentially differentiated cells in which at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the differentiated cells express at least one marker indicating mDA or an mDA precursor. Non-limiting examples of markers indicating mDA or an mDA precursor include engrailed-1 (EN1), orthodenticle homeobox 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor-associated-1 protein (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1α (LMX1A), PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2.

[0197] In some embodiments, the expression of the at least one marker indicating mDA or mDA precursor in the differentiated cells 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 at least one SMAD inhibitor.

[0198] Treatment of cells with at least one FGF activator can lead to persistent expression of EN1. EN1 is a survival factor for midbrain dopamine neurons during development and continues to play a neuroprotective and physiological role in adult midbrain dopamine neurons. Therefore, cells persistently expressing EN1 can develop into functionally mature mDA after further development and maturation. In some 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 at least one SMAD inhibitor. In some 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 some embodiments, the differentiated cells have detectable levels of EN1 expression approximately 40 days after the initial contact of the stem cells with the at least one SMAD inhibitor.

[0199] In some embodiments, the differentiated cells derived from the methods of this disclosure do not express or have low expression of at least one biomarker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.

[0200] In some embodiments, the cells are contacted with the activators and inhibitors described herein at concentrations and durations that effectively increase detectable levels of expression of at least one DA neuronal marker (e.g., EN1), for example, wherein the cells are A9 type neuronal cells.

[0201] In some embodiments, the cells are contacted with the activators and inhibitors described herein at concentrations and durations that effectively reduce the expression of SMA, SIX1, PITX2, SIM1, POU4F1, and / or PHOX2A.

[0202] 5.2.8. Differentiation of mDA precursor into mDA

[0203] In some embodiments, the cells are further contacted with DA neuron lineage-specific activators and inhibitors, such as L-glutamine, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor β (TGFβ, e.g., TGFβ3), ascorbic acid (AA), and DAPT (also known as N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethyl ethyl 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 some embodiments, the cells are exposed to 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 some embodiments, the cells are exposed to 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 some embodiments, the cells are exposed to the aforementioned DA neuron lineage-specific activators and inhibitors for approximately 4, 5, 6, 7, or 8 days.

[0204] In some embodiments, the cells are contacted with L-glutamine at concentrations between about 0.5 mM and about 5 mM, or about 1 mM and about 5 mM, or about 1.5 mM and about 2.5 mM, or about 1 mM and about 2 mM. In some embodiments, the cells are contacted with L-glutamine at a concentration of about 2 mM.

[0205] In some embodiments, the cells are contacted with BDNF at concentrations between about 5 ng / ml and about 50 ng / ml, or between about 10 ng / ml and about 50 ng / ml, or between about 10 ng / ml and about 40 ng / ml, or between about 20 ng / ml and about 50 ng / ml, or between about 20 ng / ml and about 40 ng / ml, or between about 10 ng / ml and about 30 ng / ml, or between about 10 ng / ml and about 20 ng / ml, or between about 20 ng / ml and about 30 ng / ml. In some embodiments, the cells are contacted with BDNF at a concentration of about 20 ng / ml.

[0206] In some embodiments, the cells are contacted with ascorbic acid (AA) at concentrations between about 50 nM and about 500 nM, or between about 100 nM and about 500 nM, or between about 100 nM and about 400 nM, or between about 200 nM and about 400 nM, or between about 200 nM and about 300 nM, or between about 100 nM and about 300 nM. In some embodiments, the cells are contacted with AA at a concentration of about 200 nM.

[0207] In some embodiments, the cells are contacted with GDNF at concentrations between about 5 ng / ml and about 50 ng / ml, or between about 10 ng / ml and about 50 ng / ml, or between about 10 ng / ml and about 40 ng / ml, or between about 20 ng / ml and about 50 ng / ml, or between about 20 ng / ml and about 40 ng / ml, or between about 10 ng / ml and about 30 ng / ml, or between about 10 ng / ml and about 20 ng / ml, or between about 20 ng / ml and about 30 ng / ml. In some embodiments, the cells are contacted with GDNF at a concentration of about 20 ng / ml.

[0208] In some embodiments, the cells are contacted with cAMP at concentrations between about 200 nM and about 800 nM, or between about 200 nM and about 700 nM, or between about 300 nM and about 700 nM, or between about 300 nM and about 600 nM, or between about 400 nM and about 600 nM, or between about 450 nM and about 550 nM. In some embodiments, the cells are contacted with cAMP at a concentration of about 500 nM.

[0209] In some embodiments, the cells are contacted with TGFβ3 at concentrations between about 0.01 ng / ml and about 5 ng / ml, or between about 0.1 ng / ml and about 4 ng / ml, or between about 0.5 ng / ml and about 5 ng / ml, or between about 1 ng / ml and about 3 ng / ml, or between about 1 ng / ml and about 2 ng / ml. In some embodiments, the cells are contacted with TGFβ3 at a concentration of about 1 ng / mL.

[0210] In some implementations, such as U.S. Publication No. 2015 / 0010514 (which is incorporated herein by reference in its entirety), the differentiated midbrain DA precursor is further cultured.

[0211] 5.3. Methods for isolating midbrain dopamine neurons and their precursors

[0212] This invention provides a method for isolating mDA and its precursors based on at least one or at least two surface markers. In some embodiments, the surface markers are negative surface markers, wherein the cells do not express detectable levels of the negative surface markers. In some embodiments, the cells express a reduced level of the negative surface markers compared to the average expression level of the negative surface markers in the cell population from which the cells were isolated.

[0213] In some embodiments, the surface marker is a positive surface marker, wherein the cells express a detectable level of the positive surface marker. In some embodiments, the level of the positive surface marker expressed by the cells is increased compared to the average expression level of the positive surface marker in the cell population from which the cells were isolated.

[0214] In some embodiments, the method of this disclosure for isolating mDA and its precursors from a cell population includes isolating cells that do not express at least one negative surface marker at a detectable level. In some embodiments, the method of this disclosure for isolating mDA and its precursors from a cell population includes isolating cells that do not express at least one negative surface marker at a detectable level 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 some embodiments, the method of this disclosure for isolating mDA and its precursors from a cell population includes isolating cells that express at least one positive surface marker at a detectable level. In some embodiments, the method of this disclosure for isolating mDA and its precursors from a cell population includes isolating cells that 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.

[0215] In some embodiments, the method of this disclosure for isolating mDA and its precursors from a cell population includes isolating cells that do not express at least one negative surface marker at a detectable level but express at least one positive surface marker at a detectable level. In some embodiments, the method of this disclosure for isolating mDA and its precursors from a cell population includes isolating cells that: (a) do not express at least one negative surface marker at a detectable level 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 surface marker in the cell population.

[0216] In some embodiments, the negative surface marker is selected from CD49e (also known as integrin α5), CD99, CD340, and combinations thereof. In some embodiments, the positive surface marker is selected from CD171, CD184, and combinations thereof.

[0217] In some embodiments, the method of this disclosure for isolating mDA and its precursors from a cell population includes isolating cells that do not express a detectable level of CD49e but express a detectable level of CD184. In some embodiments, the method of this disclosure for isolating mDA and its precursors from a cell population includes isolating cells that do not express a detectable level of CD49e or express a reduced level of CD49e compared to the average expression level of CD49e in the cell population; and express an increased level of CD184 compared to the average expression level of CD184 in the cell population.

[0218] Any cell separation technique based on surface markers known in the art can be used in the methods of this disclosure. In some embodiments, flow cytometry is used in the separation methods of this disclosure.

[0219] 5.4. Cell populations and compositions

[0220] This invention provides a population of cells differentiated in vitro, wherein at least about 50% (e.g., at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) of the cells express at least one marker indicating mDA or a precursor of mDA. Non-limiting examples of markers indicating mDA or a precursor of mDA include EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LMO3, SNCA, ADCAP1, CHRNA4, and GIRK2. This invention also provides compositions comprising such cell populations. In some embodiments, the in vitro differentiated cells are obtained by the differentiation methods described herein (e.g., in Section 5.2).

[0221] In some 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 differentiated cells express at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.

[0222] The present invention also provides a 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) without expressing at least one negative surface marker disclosed herein (e.g., in Section 5.3). The present invention also provides a 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 at least one positive marker in the cell population; and do not express at a detectable level of at least one negative surface marker disclosed herein (e.g., in Section 5.3) or express at a decreased level compared to the average expression level of at least one negative surface marker disclosed herein (e.g., in Section 5.3).

[0223] In some 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 detectable levels of CD49e, but express detectable levels of CD184. In some 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 detectable levels of CD49e or express CD49e at a reduced level compared to the average expression level of CD49e in the cell population; while expressing CD184 at an increased level compared to the average expression level of CD184 in the cell population. Furthermore, the present invention provides compositions comprising such cell populations.

[0224] In some embodiments, the cells are contained in a composition that also includes a biocompatible scaffold or matrix, such as a biocompatible three-dimensional scaffold that promotes tissue regeneration when the cells are implanted or transplanted into a subject. In some embodiments, the biocompatible scaffold includes extracellular matrix materials, synthetic polymers, cytokines, collagen, peptides or proteins, polysaccharides including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or hydrogels. (See, for example, U.S. Publications 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, each of which is incorporated herein by reference in its entirety). In some embodiments, the composition also includes growth factors for promoting the maturation of implanted / transplanted cells into midbrain DA cells.

[0225] In some embodiments, the composition administered to the subject contains about 1 × 10⁻⁶ 4 To approximately 1×10 10 Approximately 1×10 4 To approximately 1×10 5 Approximately 1×10 5 To approximately 1×10 9 Approximately 1×10 5 To approximately 1×10 6 Approximately 1×10 5 To approximately 1×10 7 Approximately 1×10 6 To approximately 1×10 7 Approximately 1×10 6 To approximately 1×10 8 Approximately 1×10 7 To approximately 1×10 8Approximately 1×10 8 To approximately 1×10 9 Approximately 1×10 8 To approximately 1×10 10 or approximately 1×10 9 To approximately 1×10 10 Cell populations. In some embodiments, approximately 1 × 10⁻⁶ cells are administered to the subject. 5 To approximately 1×10 7 Each of its cells.

[0226] In some embodiments, the composition is frozen. In some 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.

[0227] In some embodiments, the composition further comprises a biocompatible scaffold or matrix, such as a biocompatible three-dimensional scaffold that promotes tissue regeneration when cells are implanted or transplanted into a subject. In some embodiments, the biocompatible scaffold includes extracellular matrix materials, synthetic polymers, cytokines, collagen, peptides or proteins, polysaccharides including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or hydrogels. (See, for example, U.S. Publications 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, each of which is incorporated herein by reference in its entirety.)

[0228] In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition may be used for the prevention and / or treatment of neurodegenerative diseases, including Parkinson's disease, Huntington's disease, Alzheimer's disease, and multiple sclerosis.

[0229] This disclosure also provides an apparatus or composition comprising differentiated cells, as disclosed herein. Non-limiting examples of the apparatus include syringes, thin glass tubes, stereotactic needles, and cannulas.

[0230] 5.5. Treatment methods for neurodegenerative diseases

[0231] 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 this disclosure provides methods for treating neurodegenerative diseases. In some embodiments, the method includes administering to a subject suffering from a neurodegenerative disease an effective amount of the stem cell-derived mDA of this disclosure or a composition comprising thereof. In some embodiments, the method includes 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 some embodiments, the method includes administering to a subject with 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 a reduced level of at least one negative surface marker (e.g., CD49e) compared to the average expression of at least one positive surface marker (e.g., CD184) compared to the average expression of at least one positive surface marker (e.g., CD184) compared to the average expression of at least one positive surface marker (e.g., CD184) compared to the average expression of at least one positive surface marker (e.g., CD184) compared to the average expression of at least one positive surface marker (e.g., CD184) from a cell population isolated therefrom; or a composition comprising such cells. In some embodiments, the composition is a pharmaceutical composition that also comprises a pharmaceutically acceptable carrier.

[0232] Non-limiting examples of neurodegenerative diseases include Parkinson's disease, Huntington's disease, Alzheimer's disease, and multiple sclerosis.

[0233] In some 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; bradykinesia or slowness of movement; stiffness or pain in the limbs and trunk; postural instability; or impaired balance and coordination.

[0234] In some embodiments, the neurodegenerative disease is Parkinson's disease, which refers to a condition associated with insufficient dopamine in the basal ganglia, part of the brain that controls movement. Symptoms include tremor, bradykinesia (extremely slow movement), flexed posture, postural instability, and rigidity. Non-limiting examples of Parkinson's disease include corticobasal degeneration, Lewy body dementia, multiple system atrophy, and progressive supranuclear palsy.

[0235] The cells or composition can be systematically or directly administered or delivered to a subject to treat or prevent neurodegenerative diseases. In some embodiments, the cells or composition are injected directly into a target organ (e.g., the central nervous system (CNS) or the peripheral nervous system (PNS)). In some embodiments, the cells or composition are injected directly into the striatum.

[0236] The cells or composition can be administered in any physiologically acceptable carrier. The cells or composition can be administered via local injection, in situ (OT) injection, systemic injection, intravenous injection, or parenteral administration. In some embodiments, the cells or composition are administered via in situ (OT) injection to a subject suffering from a neurodegenerative disease.

[0237] The cells or compositions described herein can be readily provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are easier to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within a suitable viscosity range to provide a longer contact time with a specific tissue. Liquid or viscous compositions may contain a carrier, which may be a solvent or dispersion medium comprising, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. If desired, sterile injectable solutions can be prepared by adding the compositions of the subject matter of this disclosure (e.g., compositions containing stem cell-derived precursors of this disclosure) to a desired amount of a suitable solvent containing varying amounts of other components. Such compositions can be mixed with suitable carriers, diluents, or excipients (e.g., sterile water, physiological saline, glucose, dextrose, etc.). The compositions may also be lyophilized. The composition may contain excipients such as wetting agents, dispersants or emulsifiers (e.g., methylcellulose), pH buffers, gelling or thickening additives, preservatives, flavoring agents, coloring agents, etc., depending on the route of administration and the desired formulation. Reference may be made to standard texts, such as the 17th edition of “Remington’s Pharmaceutical Science” (1985), which is incorporated herein by reference, to prepare suitable formulations without excessive experimentation.

[0238] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). Prolonged absorption of injectable drug forms can be achieved by using agents with delayed absorption (e.g., alum monostearate and gelatin).

[0239] If necessary, a pharmaceutically acceptable thickener may be used to maintain the viscosity of the composition at the selected level. Methylcellulose may be used because it is readily available, economical, and easy to use. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The concentration of the thickener depends on the selected pharmaceutical agent. It is important to use an amount that achieves the selected viscosity. The selection of a suitable carrier and other additives will depend on the specific route of administration and the nature of the particular dosage form, such as a liquid dosage form (e.g., whether the composition will be formulated as a solution, suspension, gel, or other liquid form, such as a time-release or liquid-filled form).

[0240] Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert and not affect the activity or efficacy of the stem cell-derived precursors of this disclosure. This will not present any problems to those skilled in the art of chemistry and pharmaceutical principles, or problems can be easily avoided from this disclosure and the documents cited herein by referring to standard texts or by simple experiments (not involving multiple experiments).

[0241] One consideration regarding the use of the cell therapy is the number of cells required to achieve optimal results. Optimal results include, but are not limited to, reproliferation of the CNS and / or PNS regions in subjects with neurodegenerative diseases, and / or improvement in the function of the CNS and / or PNS in subjects.

[0242] An “effective dose” (or “therapeutic effective dose”) is a dose sufficient to affect a beneficial or anticipated clinical outcome following treatment. An effective dose may be administered to the subject in one or more doses. In therapeutic terms, an effective dose is a dose sufficient to reduce, improve, 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 dose is typically determined by a physician on a case-by-case basis and within the skill of a person skilled in the art. Several factors are typically considered when determining the appropriate dose to achieve an effective dose. These factors include the subject’s age, sex, and weight; the disease being treated; the severity of the disease; and the form and effective concentration of the cells administered.

[0243] In some embodiments, the effective amount of the cells is sufficient to reproliferate the CNS and / or PNS regions of a subject suffering from a neurodegenerative disease. In some embodiments, the effective amount of the cells is sufficient to improve the function of the CNS and / or PNS of a subject suffering from a neurodegenerative disease; for example, the improved function may be about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the CNS and / or PNS function of a normal person.

[0244] The number of cells to be administered varies depending on the subject being treated. In some embodiments, the subject is administered a dose ranging from approximately 1 × 10⁻⁶ cells. 4 To approximately 1×10 10 From approximately 1×10 4 To approximately 1×10 5 From approximately 1×10 5 To approximately 1×10 9 From approximately 1×10 5 To approximately 1×10 6 From approximately 1×10 5 To approximately 1×10 7 From approximately 1×10 6 To approximately 1×10 7 From approximately 1×10 6 To approximately 1×10 8 From approximately 1×10 7 To approximately 1×10 8 From approximately 1×10 8 To approximately 1×10 9 From approximately 1×10 8 To approximately 1×10 10 or from approximately 1×10 9 To approximately 1×10 10 The cells described. In some embodiments, approximately 1 × 10... 5 To approximately 1×10 7 The cells are administered to a subject suffering from a neurodegenerative disease. In some embodiments, approximately 1 × 102 cells are used. 6 To approximately 1×10 7 The cells are administered to a subject suffering from a neurodegenerative disease. In some embodiments, approximately 1 × 102 cells are used. 6 To approximately 4×10 6 The cells described are administered to subjects suffering from neurodegenerative diseases. The precise determination of the effective dose may be based on individual factors of each subject, including their size, age, sex, weight, and the specific condition of the subject. Those skilled in the art can readily determine the dose according to the present invention and their knowledge in the art.

[0245] 5.6. Reagent Kit

[0246] This disclosure provides a kit for inducing stem cell differentiation into mDA or its precursor. In some 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 some embodiments, the kit further comprises (e) instructions for inducing stem cells to differentiate into a differentiated cell population expressing at least one marker indicating mDA or its precursor.

[0247] In some embodiments, the description includes contacting stem cells with inhibitors, activators, and molecules in a specific order. The order of contact with inhibitors, activators, and molecules can be determined by the cell culture medium used to culture the stem cells.

[0248] In some embodiments, the description includes contacting the stem cells with the inhibitors, activators, and molecules described in this disclosure (see Section 5.2).

[0249] In some embodiments, this disclosure provides kits comprising an effective amount of a unit dosage form of the cell populations or compositions disclosed herein. In some embodiments, the kits comprise sterile containers containing a therapeutic composition; these containers may be boxes, ampoules, bottles, vials, tubes, pouches, bags, blister packs, or other suitable container forms known in the art. These containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding the drug.

[0250] In some embodiments, the kit includes instructions for administering the cell population or composition to a subject suffering from a neurodegenerative disease. The instructions may include information regarding the use of the cells or composition to treat or prevent neurodegenerative diseases. In some embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosage table and administration for the treatment or prevention of neurodegenerative diseases or their symptoms; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if any), applied as a label to the container, or provided as a separate sheet of paper, brochure, card, or folder with the container.

[0251] 6. Example

[0252] The subject matter of this disclosure will be better understood by referring to the following examples, which are provided as examples and not as limitations.

[0253] Example 1: Optimization of the differentiation scheme for midbrain DA neurons

[0254] Midbrain dopamine neurons and their precursors were derived from stem cells using the previously disclosed Wnt-Boost protocol (International Publication No. WO 2016 / 196661, "7.5 μM bump protocol (modification of GMP V2B)," which is incorporated herein by reference in its entirety) (hereinafter referred to as the "Wnt-Boost protocol" or "Boost protocol"). The study found that EN1 expression decreased starting from day 11 of differentiation using the Wnt-Boost protocol (see...). Figure 1Maintaining EN1 expression in differentiating cells is crucial for the generation of mature and functional midbrain dopamine neurons. Therefore, to maintain EN1 expression, this example tested the addition of FGF8b treatment to the Wnt-Boost protocol during late differentiation (see [link to Wnt-Boost protocol]). Figure 2 In addition to the Wnt-Boost regimen, cells exposed to FGF8b from day 9 to day 16, day 10 to day 16, day 11 to day 16, day 12 to day 16, day 13 to day 16, day 14 to day 16, or day 15 to day 16 were also tested. Immunostaining on day 16 of differentiation showed that EN1 protein expression was maintained in a duration-dependent manner depending on the FGF8b exposure (see [link to relevant documentation]). Figure 3 EN1-positive cells also expressed FOXA2 and LMX1A. RNA expression measured in cells on day 30 of differentiation indicated that the expression levels of mDA or mDA precursor markers FOXA2, NURR1, LMX1A, OXT2, and TH were comparable under all conditions, and that EN1 mRNA expression was maintained in a duration-dependent manner dependent on FGF8b exposure (see [link to relevant documentation]). Figures 4A-4B However, the mRNA levels of pollution biomarkers (non-mDA or non-mDA precursor biomarkers) (e.g., SMA and SIX1) are also induced in a duration-dependent manner by FGF8b exposure (see [link to FGF8b exposure report]). Figure 4B On day 30 of differentiation, SIX1 immunostaining of cells confirmed the mRNA results (see [link to original text]). Figure 5 ).

[0255] FGF8b has been used to derive midbrain dopamine neurons from pluripotent stem cells. FGF8, FGF17, and FGF18 are subfamilies of FGF, and FGF17b and FGF18 have been shown to 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 6-OHDA-induced midbrain dopamine neuron damage (Guo et al., Neuroscience, 2017 Jul 25; 356:229-241). Furthermore, FGF8b (isthmus and rhomboid ganglion 1) extends tissues along the connections between the induced Gbx2 domain and the remaining Otx2 region in the midbrain. FGF8a, FGF17, and FGF18 are responsible for midbrain expansion and the upregulation of midbrain genes. FGF8b, FGF17, and FGF18 are all the same FGF subgroup that paracrine FGFs to FGF8b.

[0256] Under the WNT-Boost protocol, FGF8b, FGF17, and FGF18 were tested from day 12 to day 16 by adding them to cell cultures at a concentration of 100 ng / ml. The study found that in cells differentiated on day 16, FGF18 induced EN1 mRNA expression levels similar to FGF8b, but SMA mRNA expression levels were decreased (see [link to study]). Figure 6 and Figure 7 Similar to FGF8b, EN1 protein expression was also highly maintained in a manner dependent on the duration of FGF18 contact.

[0257] During the maturation stage of differentiation, EN1 remained highly maintained under FGF8b and FGF18 treatment (see...). Figure 8 Furthermore, compared to cells differentiated via the WNT-Boost protocol, cells treated with FGF18 and FGF8b showed decreased PITX2 expression levels, while cells treated with FGF18 showed lower SMA1 and SIX1 expression levels than those treated with FGF8b (see [link to relevant documentation]). Figure 8 These results indicate that, compared with FGF8b treatment, FGF18 treatment resulted in sustained EN1 expression while minimizing or reducing the expression levels of non-mDA biomarkers.

[0258] The in vivo viability of differentiated cells generated by the WNT-boost+FGF18 regimen was assessed. Cells generated by both the WNT-boost and WNT-boost+FGF18 regimens were transplanted into intact mouse protein. Compared to cells generated by the WNT-boost regimen, cells generated by the WNT-boost+FGF18 regimen maintained improved EN1 expression in vivo. Figure 35A Cells generated by the WNTboost+FGF18 regimen showed better striatal innervation one month post-transplantation, with fibers extending from the graft core to the periphery. Figure 35B ).

[0259] Example 2: Purification of mDA using reporter

[0260] NURR1 is a marker of post-mitotic and immature midbrain dopamine neurons, and is also expressed in mature midbrain dopamine neurons. It is a transcription factor that contributes to the differentiation and maintenance of dopamine neurons.

[0261] To purify mDA from the cell population, this example uses the endogenous NURR1::GFP reporter hPSC (see [link to example]). Figure 9A mDA was differentiated from reporter cell lines. GFP-positive cells were isolated on day 25 of differentiation based on FASC, following high induction of MURR1 mRNA expression from day 20 of differentiation (see [link to FASC]). Figures 9B-9C ).

[0262] Single-cell qRT-PCR was performed on NURR1:GFP-positive cells isolated at days 25 and 40 of differentiation. It was found that at day 40 of differentiation, nearly 100% of NURR1:GFP-positive cells expressed TH (a mature mDA marker), FOXA2, and LMX1A, indicating mDA fate (see [link to relevant documentation]). Figure 10A The isolated NURR1:GFP-positive cells were cultured for up to day 60, and the results showed that these cells expressed high levels of TH, indicating that these cells were high-purity mDA (see...). Figure 10B ).

[0263] On 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. Neural processes were observed growing in the cell transplantation area (see...). Figure 11 ).

[0264] NURR1:GFP hPSCs were cultured under WNT-Boost and WNT-Boost+FGF18 protocols (days 12–16). NURR1:GFP-positive cells were isolated on day 25 of differentiation and then continuously cultured until day 40. On day 40 of differentiation, these midbrain DA neurons expressed high levels of TH and FOXA2 (see [link to relevant documentation]). Figure 12 ).

[0265] mRNA expression analysis showed that mDA derived from the WNT-Boost+FGF18 (days 12-16) protocol and sorted by NURR1:GFP had higher EN1 expression levels than mDA derived from WNT-Boost and sorted by NURR1:GFP (see [link to mRNA expression analysis]). Figure 13A These sorted cells were transplanted into immunodeficient mice. Both mDAs derived from the WNT-Boost and WNT-Boost+FGF18 (days 12-16) regimens showed excellent cell viability and expressed the biomarkers SC121 and TH. However, cells treated with FGF18 (WNT-Boost+FGF18 regimen) showed better neurite growth in the transplantation area (see [link to article]). Figure 13B ).

[0266] Example 3: Surface markers found in purified DA neurons

[0267] Published research indicates that each iPSC line varies due to induction by specific cell types. It is difficult to generate reporter lines for purifying midbrain DA cells from each iPSC line. Furthermore, genetically engineered cells are not suitable for clinical use. This embodiment uses the NURR1::GFP reporter cell line to identify candidate surface markers, particularly those enriched in NURR1::GFP-positive cells but not in NURR1::GFP-negative cells, and vice versa.

[0268] In this embodiment, 387 surface markers derived from NURR1:GFP hPSC in mDA-differentiated cells were tested on day 25 of differentiation (see [link to documentation]). Figure 14 ).

[0269] Two positive CD markers, CD171 and CD184, were enriched in the NURR1::GFP positive population (see...). Figures 15A-15B Three negative CD markers, CD49e, 99, and 340, were enriched in the NURR1::GFP negative population (see [link to relevant documentation]). Figures 16A-16B ).

[0270] Cells were sorted for CD49e (negative and / or weakly expressed) on day 25 of differentiation under either the WNT-Boost protocol or the WNT-Boost+FGF18 protocol (days 12-16) and cultured for another 10 days. Cell morphology showed that these cells were essentially pure mDA (see [link to relevant documentation]). Figure 17 mDA separated on day 40 of differentiation (sorted on day 25 and cultured for another 15 days) showed high TH immunostaining ( Figure 18 ).

[0271] CD49e markers were 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 with CD49e (negative and / or weakly expressed) and cultured for another 15 days (day 40 of differentiation) under the WNT-boost protocol and the WNT-boost+FGF18 (days 12–16) protocol (see [link to relevant documentation]). Figures 19-20 These sorted mDA samples exhibit high TH immunostaining (see...). Figure 21 ).

[0272] mRNA expression analysis showed that CD49e sorted cells differentiated under the WNT-boost+FGF18 protocol (days 12–16) had higher EN1 expression levels and lower PITX2 (a glutamatergic neuron marker (hypothalamic nucleus marker)) expression levels compared to CD49e sorted cells differentiated under the WNT-boost protocol. Sorted cells differentiated under both protocols showed little or no expression of non-midbrain DA markers (HOXA2, SMA1, and SIX1) (see [link to relevant documentation]). Figure 22 ).

[0273] All three negative CD markers, CD49e, CD99, and CD340, were detected. On day 25 of differentiation and for an additional 15 days of culture (day 40 of differentiation), substantially pure neuronal morphology was observed in cells sorted with CD49e, CD99, or CD340 (negative and / or weakly expressed cells) (see [link to study]). Figure 23 However, mRNA expression in sorted cells showed increased expression of non-DA neuronal markers (including PHOX2A, PITX2, POU4F1, and SIM1), suggesting that separation based on CD49e, CD99, or CD340 does not exclude non-DA neurons. Figure 24 ).

[0274] It is believed that a dual sorting strategy using CD184 can address the deficiencies of negative CD markers. Cxcr4 (CD184) plays a crucial role in the migration and orientation of midbrain dopamine (DA) neurons during mouse midbrain development. FGF18-treated mDA is then sorted via CD184. + / CD49e - After sorting, the A9 midbrain DA subtype can be enriched.

[0275] On day 25 of differentiation, midbrain DA cells derived from NURR1::GFP hPSCs were subjected to FACS sorting using CD49E (PE) and CD171 (APC). Results showed that individual CD49e-negative sorted cells contained approximately 63% of the NURR1::GFP fraction. CD171-positive sorting did not enrich the NURR1::GFP population bound to CD49e (see [link to FACS]). Figure 25 ).

[0276] However, sorting with both CD49e and CD184 (positive cells) could enrich NURR1:GFP to approximately 80% compared to sorting cells alone (CD49e; 63%) (see [link to article]). Figure 26 ).

[0277] Then, on day 25 of differentiation, cells were sorted using FACS by CD49e and CD171 or CD49e and CD184. Cells cultured on day 2 post-sorting showed pure neuronal morphology, except for sorted cells based on higher CD49e levels (see [link to FACS]). Figure 27 ).

[0278] mRNA expression showed that, compared to cells sorted by other methods, CD49e... - / CD184 + (CD49e-negative / CD184-positive) cells showed high expression levels of mDA markers (NURR1, EN1, PITX3) and low expression levels of non-mDA markers (PITX2, SIM1, and POU4F1) (see [link to relevant documentation]). Figure 28 ).

[0279] Next, the reliability of CD49e and CD184 in sorting mDA was investigated. For example... Figure 29 As shown, in in vitro differentiated cells, single CD49e - Sorting enriched the NURR1:GFP-positive cell population from ~20% to as high as 43%. For example... Figure 30 As shown, in in vitro differentiated cells, double CD49e - and CD184 + The sorting enriched the NURR1::GFP positive fraction to 74% and 85%.

[0280] After two weeks of in vitro culture, sorted cells showed high TH levels co-expressing FOXA2 and GFP. + mDA, which confirms the properties of mDA (see mDA). Figure 31 ).

[0281] mRNA expression showed that, compared with other CD-sorted cells, cells sorted by dual CD markers (CD49e) showed higher mRNA expression levels. - and CD184 + mDA marker expression levels are typically higher on day 40 of differentiation (see [link]). Figure 32 ), while the expression levels of non-mDA markers were lower (see Figure 33 ).

[0282] The in vivo viability of differentiated cells sorted using the novel surface markers disclosed herein was assessed. Differentiated cells were generated according to the WNT-boost protocol, and cells with weak CD49e and high CD184 were sorted and transplanted into intact mouse brains. Compared to unsorted cells, tissue transplanted with sorted cells was rich in Th+ cells (…). Figure 34A One month after transplantation, the number of SOX2+ precursors and KI67+ dividing cells decreased. Figure 34B-3 4D).

[0283] Although the subject matter and advantages of this disclosure have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of this disclosure. Furthermore, the scope of this application is not limited to the specific embodiments of the processes, machines, manufactures, and material compositions, means, methods, and steps described in the specification. Those skilled in the art will readily understand from this invention the subject matter, processes, machines, manufactures, material compositions, means, methods, or steps of this disclosure (existing or to be developed thereafter that can perform substantially the same function or achieve substantially the same results based on the subject matter of this disclosure and the corresponding embodiments described herein). Therefore, the appended claims are intended to encompass such processes, machines, manufactures, material compositions, means, methods, or steps within their scope.

[0284] Various patents, patent publications, publications, product descriptions, schemes and serial numbers are cited in this application, and for all purposes, the entire contents of which are incorporated herein by reference.

Claims

1. An in vitro method for inducing differentiation of a cell expressing forkhead box protein A2 (FOXA2) and LIM homeobox transcription factor 1 alpha (LMX1A), comprising contacting the cell 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 a midbrain dopamine neuron (mDA) or a precursor thereof, wherein the at least one activator of FGF signaling is selected from the group consisting of FGF18, FGF17, and combinations thereof.

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

3. The method of claim 1, wherein the cell is contacted with the at least one activator of FGF signaling for at most 15 days.

4. The method of claim 1, wherein the cell is contacted with the at least one activator of FGF signaling for 5 days.

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

6. The method of claim 1, wherein 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, PITX3, LMO3, SNCA, ADCAPl, CHRNA4, GIRK2, and combinations thereof.

7. The method of claim 1, wherein 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, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.

8. The method of claim 1, further comprising placing the population of differentiated cells under conditions conducive to differentiation of midbrain dopamine neuron precursors to midbrain dopamine neurons.

9. The method of claim 8, wherein the conditions comprise exposing the cells to at least one of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), cyclic adenosine monophosphate (cAMP), transforming growth factor beta 3 (TGFp3), ascorbic acid (AA), and DAPT.

10. The method of claim 1, wherein the 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 recombinant pluripotent cell.

11. The method of claim 1, wherein the cell is a human stem cell.

12. The method of claim 1, wherein the cell is a pluripotent or multipotent stem cell.

13. The method of claim 12, wherein the cell is a pluripotent stem cell.

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

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

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

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

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