Method for inducing differentiation of pluripotent stem cells into dopaminergic neural cells , method for producing cell group containing dopaminergic neural cells, and cell group containing dopaminergic neural cells which is produced by said method

Abstract

The present invention provides a method for inducing the differentiation of pluripotent stem cells into dopaminergic neural cells or a method for producing a cell group containing dopaminergic neural cells from pluripotent stem cells, each of the methods comprising: (1) bringing the pluripotent stem cells into contact with a TGFβ inhibitor, a BMP inhibitor, and / or a GSK3β inhibitor; and (2) expressing Ascl1 gene in the pluripotent stem cells.

Description

Method for inducing differentiation of dopaminergic neurons from pluripotent stem cells, method for producing a cell population containing dopaminergic neurons, and a cell population containing dopaminergic neurons produced thereby

[0001] The present invention relates to a method for inducing differentiation of dopamine neurons from pluripotent stem cells, a method for producing a cell population containing dopamine neurons, and a cell population containing dopamine neurons produced thereby.

[0002] Since the discovery of pluripotent cells (pluripotent stem cells), such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), which are obtained by introducing undifferentiated cell-specific genes into somatic cells, clinical research has progressed rapidly in the field of regenerative medicine, and in the field of drug discovery, research has progressed rapidly in which pluripotent stem cells are differentiated into cells of desired tissues, thereby exploring the causes of disease and using them to screen new therapeutic drugs.

[0003] Pluripotent stem cells can be induced to differentiate into various somatic cells that constitute the body, and many methods for inducing differentiation into desired cells have been reported. For example, methods for inducing differentiation into neurons have been reported for the purpose of research and treatment in the field of the central nervous system. For example, a method for differentiating human pluripotent stem cells into neurons using the TGFβ inhibitor SB431542, the BMP inhibitor Dorsomorphine, and the GSK3β inhibitor CHIR99021 has been reported (Patent Document 1). In addition, a method for differentiating mouse and human embryonic stem cells into dopaminergic neurons by expressing the Lmx1a gene, the Nurr1 gene, and the Ascl1 gene has been reported (Non-Patent Document 1).

[0004] As mentioned above, methods for inducing differentiation of dopaminergic neurons have been disclosed. However, these methods have drawbacks, such as taking a long time (21 days or more) and resulting in a high rate of dead cells, and therefore, their application to disease research has not progressed to date.

[0005] International Publication No. 2017 / 170328

[0006] Yi Han Ng, et al. , Stem Cell Reports. 2021 Jul 13;16(7):1763-1776

[0007] An object of the present invention is to provide a novel method for efficiently inducing differentiation of pluripotent stem cells into dopaminergic neurons.

[0008] As a result of extensive research to solve the above-mentioned problems, the inventors discovered that dopaminergic neurons can be efficiently generated by inducing the expression of the Ascl gene in pluripotent stem cells that have been contacted with a TGFβ inhibitor and / or a BMP inhibitor and / or a GSK3β inhibitor, and thus completed the present invention.

[0009] That is, the gist of the present invention relates to, for example, the following: [1] A method for inducing differentiation of dopaminergic neurons from pluripotent stem cells, comprising: (1) contacting the pluripotent stem cells with a TGFβ inhibitor, a BMP inhibitor, and / or a GSK3β inhibitor, and (2) expressing an Ascl1 gene in the pluripotent stem cells. [2] The method according to item 1, wherein step (1) is: (1) contacting the pluripotent stem cells with a TGFβ inhibitor, a BMP inhibitor, and a GSK3β inhibitor. [3] The method according to item 1 or 2, wherein the TGFβ inhibitor is SB431542. [4] The method according to item 1 or 2, wherein the BMP inhibitor is dorsomorphin. [5] The method according to item 1 or 2, wherein the GSK3β inhibitor is CHIR99021. [6] The method according to any one of items 1 to 5, further comprising expressing one or more genes selected from the group consisting of Lmx1A gene, FoxA2 gene, Nurr1 gene, Pitx3 gene, and En1 gene in the pluripotent stem cells. [7] The method according to any one of items 1 to 6, wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells).

[0010] [8] A method for producing a cell population containing dopamine neurons from pluripotent stem cells, comprising: (1) contacting the pluripotent stem cells with a TGFβ inhibitor, a BMP inhibitor, and / or a GSK3β inhibitor, and (2) expressing the Ascl1 gene in the pluripotent stem cells. [9] The method according to Item 8, wherein step (1) is: (1) contacting the pluripotent stem cells with a TGFβ inhibitor, a BMP inhibitor, and a GSK3β inhibitor.

[10] The method according to Item 8 or 9, wherein the TGFβ inhibitor is SB431542.

[11] The method according to Item 8 or 9, wherein the BMP inhibitor is dorsomorphin.

[12] The method according to Item 8 or 9, wherein the GSK3β inhibitor is CHIR99021.

[13] The method according to any one of items 8 to 12, further comprising expressing one or more genes selected from the group consisting of Lmx1A gene, FoxA2 gene, Nurr1 gene, Pitx3 gene, and En1 gene in the pluripotent stem cells.

[14] The method according to any one of items 8 to 13, wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells).

[15] A cell population comprising dopamine neurons produced by the method according to any one of items 8 to 14.

[0011] According to the present invention, a method for increasing the efficiency of inducing differentiation from pluripotent stem cells into dopaminergic neurons is provided, and the present invention also shortens the time required to induce differentiation from pluripotent stem cells into dopaminergic neurons compared to conventional methods.

[0012] FIG. 1A shows a scheme for gene transfer into iPS cells in an example. FIG. 1B shows a scheme for differentiation of iPS cells into dopaminergic neurons in an example. SB: SB431542, DM: Dorsomerphin, CHIR: CHIR 99021. FIG. 1C shows the gene expression vectors used in the example. FIG. 2 shows microscopic images of iPS cells after induction of differentiation in which the ASCL1 gene was induced after non-DSC treatment (DSC-) and DSC treatment (DSC+) in Example 5. FIG. 3 shows fluorescent microscopic images of iPS cells after induction of differentiation in which the ASCL1 gene was induced after non-DSC treatment (DSC-) and DSC treatment (DSC+) in Example 6. Blue: Cell nuclei (Hoechst (Ho)), green: βIII-tubulin (Tubb3) (neuronal cell marker) positivity. Figure 4A is a fluorescence microscope image of iPS cells after differentiation induction in which the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene were induced after DSC-untreated (DSC-) and DSC-treated (DSC+) in Example 7. Blue: Cell nuclei (Hoechst (Ho)), red: tyrosine hydroxylase (TH) (dopamine neuron marker) positivity, green: βIII-tubulin (Tubb3) (neuronal cell marker) positivity. Figure 4B is a fluorescence microscope image of iPS cells after differentiation induction in which the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene were induced after DSC-untreated (DSC-) and DSC-treated (DSC+) in Example 7. 4A and 4B are graphs showing the percentages of βIII-tubulin (Tubb3)-positive cells and tyrosine hydroxylase (TH)-positive cells (dopamine neuron marker)-positive cells relative to the total number of cells, calculated from fluorescence microscopy images of iPS cells after differentiation induction in which the ASCL1 gene, EN1 gene, LMX1A gene, and PITX3 gene were induced (Figure 4A). Nucleus-positive cells were calculated as the total number of cells (=100%). Figure 5 shows the results of quantitative PCR analysis of the mRNA expression level of a peripheral neuron marker (PRPH) in iPS cells in which the ASCL1 gene was induced (denoted as "A") or in iPS cells in which the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene were induced (denoted as "A+5Fs") in Example 8.Figure 6A shows the results of transcriptome analysis by RNA-seq for iPS cells in which the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene were induced when non-DSC-treated (DSC-) and DSC-treated (DSC+) in Example 9. Figure 6B shows the results of transcriptome analysis by RNA-seq for iPS cells in which the ASCL1 gene was induced (denoted as "A") or iPS cells in which the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene were induced (denoted as "A+5Fs") in Example 9, when DSC-treated (DSC+). Figure 6C shows the results of transcriptome analysis by RNA-seq when iPS cells in which the ASCL1 gene was induced (denoted as "A") or iPS cells in which the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene were induced (denoted as "A+5Fs") in Example 9 were subjected to DSC treatment (DSC+). FIG. 7 shows the results of measuring the intracellular dopamine levels using HPLC-ECD in iPS cells introduced with the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, or PITX3 gene that had been subjected to DSC treatment (DSC+) and in cells in which expression of these genes had been induced with doxycycline (denoted as "iDA") or in cells that were not treated with doxycycline (denoted as "iPS(-dox)") in Example 10. DA: dopamine, DOPAC: dihydroxyphenylacetic acid, HVA: homovanillic acid, N.D.: below detection limit.

[0013] The present invention will be described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments and can be embodied in any form without departing from the spirit of the present invention.

[0014] One embodiment of the present invention provides a method for inducing differentiation of dopamine neurons from pluripotent stem cells, comprising: (1) contacting the pluripotent stem cells with a TGFβ inhibitor, a BMP inhibitor, and / or a GSK3β inhibitor; and (2) expressing the Ascl1 gene in the pluripotent stem cells.

[0015] One embodiment of the present invention provides a method for producing a cell population containing dopamine neurons from pluripotent stem cells, comprising: (1) contacting the pluripotent stem cells with a TGFβ inhibitor, a BMP inhibitor, and / or a GSK3β inhibitor; and (2) expressing the Ascl1 gene in the pluripotent stem cells. The method of the present invention has a higher efficiency of inducing dopamine neurons from pluripotent stem cells than conventional methods. Therefore, the cell population containing dopamine neurons provided by the method of the present invention contains a high proportion of dopamine neurons.

[0016] Furthermore, one embodiment of the present invention provides a cell population containing dopamine neurons produced by the above-mentioned method for producing a cell population containing dopamine neurons.

[0017] As used herein, "pluripotent stem cells" refer to stem cells that have pluripotency and can differentiate into various cells present in a living organism, and also have the ability to proliferate. Examples of such cells include, but are not limited to, embryonic stem cells (ES cells), cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, spermatogonial stem cells (GS cells), epiblast cells, embryonic germ cells (EG cells), multipotent germline stem cells (mGS cells), and induced pluripotent stem cells (iPS cells). Of these, ES cells, ntES cells, and iPS cells are preferred, with ES cells and iPS cells being more preferred, and iPS cells being particularly preferred.

[0018] The organisms from which somatic cells or pluripotent stem cells that can be used in the present invention are derived are not particularly limited, and examples include humans and non-human animals (e.g., monkeys, sheep, cows, horses, dogs, cats, rabbits, rats, mice, etc.), preferably humans.

[0019] In the present invention, human ES cell lines that can be used as pluripotent stem cells are not particularly limited, but examples include H1, H9, Shef6, khES-1, khES-2, khES-3, khES-4, and khES-5.

[0020] In the present invention, human iPS cell lines that can be used as pluripotent stem cells are not particularly limited, but examples include WD39 (fibroblast-derived), aTKA4 (T cell-derived), 201B6, 201B7, 253G1, 253G4, and 414C2 lines.

[0021] In the present invention, pluripotent stem cells may be cells obtained by introducing known reprogramming factors (reprogramming factors) into any somatic cells other than the established cell lines described above. Examples of reprogramming factors include genes such as Oct3 / 4, Klf4, c-Myc, Sox2, Nanog, Klf2, L-Myc, N-Myc, Klf5, Lin28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, β-catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Sox15, Fthl17, Sall4, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, and E-cadherin. Here, two or more genes can be selected from this gene group and introduced in any combination. Among these, a combination of Oct3 / 4, Klf4, c-Myc, Sox2, Nanog, and Klf2 is preferred. Furthermore, it is preferable that the species of the gene to be introduced is the same as the species of the cell into which it is to be introduced. For example, genes introduced into human-derived cells are preferably human genes. For example, genes introduced into human-derived somatic cells, human ES cells, or human iPS cells are preferably a combination of human Nanog (NANOG) and human Klf2 (KLF2), and more preferably a combination of human Oct3 / 4 (OCT3 / 4), human Klf4 (KLF4), human c-Myc (c-MYC), human Sox2 (SOX2), human Nanog (NANOG), and human Klf2 (KLF2).

[0022] In one embodiment of the present invention, the method for expressing the Ascl1 gene in pluripotent stem cells may be, for example, a method for expressing an endogenous Ascl1 gene, or a method for expressing the Ascl1 gene by introducing a vector having the Ascl1 gene operably linked to a known promoter capable of regulating the on / off of gene expression by any method. The Ascl1 gene applicable in the present invention may be any gene capable of expressing a functional ASCL1 protein, and may have substitutions, deletions, and / or insertions in its nucleic acid sequence or amino acid sequence relative to a reference sequence, so long as it is capable of expressing a functional ASCL1 protein. For example, the nucleic acid sequence of the human Ascl1 (ASCL1) gene may be a nucleic acid sequence corresponding to the nucleic acid sequence (SEQ ID NO: 1) of human Ascl1 (ASCL1) mRNA registered in the GenBank database as NM_004316.4 or a part thereof, or may be a nucleic acid sequence corresponding to the open reading frame (ORF) of the gene, or may be a nucleic acid sequence having 85% or more, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, sequence identity with these nucleic acid sequences. Furthermore, the nucleic acid sequence of the human Ascl1 (ASCL1) gene may be, for example, a nucleic acid sequence encoding the amino acid sequence (SEQ ID NO: 2) registered in the GenPept database as NP_004307.2, or a nucleic acid sequence encoding a protein having an amino acid sequence that has 85% or more, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with this amino acid sequence.

[0023] As used herein, a "functional" protein refers to a protein that exhibits a function equivalent to that of a wild-type protein. For example, if the protein is a transcription activator, the protein exhibits at least 50% or more, for example, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the transcription activity exhibited by the wild-type transcription activator.

[0024] In one embodiment of the present invention, in addition to the Ascl1 gene, it is preferable to further express one or more genes selected from the group consisting of the Lmx1A gene, FoxA2 gene, Nurr1 gene (also referred to as the "NR4A2 gene"), Pitx3 gene, and En1 gene, which express transcription factors. It is more preferable to express the Ascl1 gene, Lmx1A gene, FoxA2 gene, Nurr1 gene, Pitx3 gene, and En1 gene. By expressing the above transcription factors in addition to the Ascl1 gene, dopamine neurons are more efficiently induced. The method for expressing the transcription factor may be, for example, a method for expressing a gene encoding an endogenous transcription factor, or a method for expressing the gene by introducing a vector having a gene encoding the transcription factor operably linked to a known promoter capable of regulating the on / off of gene expression by any method. Furthermore, the Ascl1 gene and the genes encoding the above-mentioned transcription factors may be incorporated into the same vector, or into separate vectors, or two, three, four, five, or six of the above may be incorporated into the same vector.

[0025] The Lmx1A gene applicable to the present invention may be any gene capable of expressing a functional LMX1A protein, and as long as it is capable of expressing a functional LMX1A protein, its nucleic acid sequence or amino acid sequence may have substitutions, deletions, and / or insertions relative to a reference sequence. For example, the nucleic acid sequence of the human Lmx1A (LMX1A) gene may be the nucleic acid sequence of human Lmx1A (LMX1A) mRNA registered in the GenBank database as NM_177398.4 (SEQ ID NO: 3) or a portion thereof, or may be a nucleic acid sequence corresponding to the open reading frame (ORF) of the gene, or may be a nucleic acid sequence having 85% or more, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, sequence identity with these nucleic acid sequences. Furthermore, the nucleic acid sequence of the human Lmx1A (LMX1A) gene may be, for example, a nucleic acid sequence encoding the amino acid sequence (SEQ ID NO: 4) registered in the GenPept database as NP_796372.1, or may be a nucleic acid sequence encoding a protein having an amino acid sequence that has 85% or more, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, sequence identity with this amino acid sequence.

[0026] The FoxA2 gene applicable to the present invention may be any gene capable of expressing a functional FOXA2 protein, and may have substitutions, deletions, and / or insertions in its nucleic acid or amino acid sequence relative to a reference sequence, as long as it is capable of expressing a functional FOXA2 protein. For example, the nucleic acid sequence of the human FoxA2 (FOXA2) gene may be the nucleic acid sequence of human FoxA2 (FOXA2) mRNA registered in the GenBank database as NM_021784.5 (SEQ ID NO: 5) or a portion thereof, or may be a nucleic acid sequence corresponding to the open reading frame (ORF) of the gene, or may have 85% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, sequence identity to these nucleic acid sequences. The nucleic acid sequence of the human FoxA2 (FOXA2) gene may be, for example, a nucleic acid sequence encoding the amino acid sequence (SEQ ID NO: 6) registered in the GenBank database as NP_068556.2, or a nucleic acid sequence encoding a protein having an amino acid sequence that shares 85% or more, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, sequence identity with this amino acid sequence.

[0027] The Nurr1 gene applicable to the present invention may be any gene capable of expressing a functional NR4A2 protein, and as long as it is capable of expressing a functional NR4A2 protein, its nucleic acid sequence or amino acid sequence may have substitutions, deletions, and / or insertions relative to a reference sequence. For example, the nucleic acid sequence of the human Nurr1 (NR4A2) gene may be the nucleic acid sequence of the human Nurr1 (NR4A2) mRNA registered in the GenBank database as NM_006186.4 (SEQ ID NO: 7) or a portion thereof, or may be a nucleic acid sequence corresponding to the open reading frame (ORF) of the gene, or may be a nucleic acid sequence having 85% or more, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, sequence identity with these nucleic acid sequences. Furthermore, the nucleic acid sequence of the human Nurr1 (NR4A2) gene may be, for example, a nucleic acid sequence encoding the amino acid sequence (SEQ ID NO: 8) registered in the GenPept database as NP_006177.1, or a nucleic acid sequence encoding a protein having an amino acid sequence that has 85% or more, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, sequence identity with this amino acid sequence.

[0028] The Pitx3 gene applicable to the present invention may be any gene capable of expressing a functional PITX3 protein, and as long as it is capable of expressing a functional PITX3 protein, the nucleic acid sequence or amino acid sequence may have substitutions, deletions, and / or insertions relative to a reference sequence. For example, the nucleic acid sequence of the human Pitx3 (PITX3) gene may be the nucleic acid sequence of human Pitx3 (PITX3) mRNA registered in the GenBank database as NM_005029.4 (SEQ ID NO: 9) or a portion thereof, or may be a nucleic acid sequence corresponding to the open reading frame (ORF) of the gene, or may be a nucleic acid sequence having 85% or more, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, sequence identity with these nucleic acid sequences. Furthermore, the nucleic acid sequence of the human Pitx3 (PITX3) gene may be, for example, a nucleic acid sequence encoding the amino acid sequence (SEQ ID NO: 10) registered in the GenPept database as NP_005020.1, or a nucleic acid sequence encoding a protein having an amino acid sequence that has 85% or more, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, sequence identity with this amino acid sequence.

[0029] The En1 gene applicable to the present invention may be any gene capable of expressing a functional EN1 protein, and as long as it is capable of expressing a functional EN1 protein, the gene may have substitutions, deletions, and / or insertions in its nucleic acid or amino acid sequence relative to a reference sequence. For example, the nucleic acid sequence of the human En1 (EN1) gene may be a nucleic acid sequence corresponding to the nucleic acid sequence (SEQ ID NO: 11) of human En1 (EN1) mRNA registered in the GenBank database as NM_001426.4 or a part thereof, or a nucleic acid sequence corresponding to the open reading frame (ORF) of the gene, or a nucleic acid sequence having 85% or more, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, sequence identity to these nucleic acid sequences. Furthermore, the nucleic acid sequence of the human En1 (EN1) gene may be, for example, a nucleic acid sequence encoding the amino acid sequence (SEQ ID NO: 12) registered in the GenPept database as NP_001417.3, or a nucleic acid sequence encoding a protein having an amino acid sequence that has 85% or more, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with this amino acid sequence.

[0030] The above-mentioned genes applicable to the present invention may be derived from any organism, but are preferably derived from mammals (e.g., humans, non-human primates, rodents (e.g., mice, rats, hamsters, guinea pigs, etc.), rabbits, dogs, cows, horses, pigs, cats, goats, sheep, etc.), more preferably from humans and non-human primates, and particularly preferably from humans.

[0031] The gene expression vector that can be used in the present invention is not particularly limited, but for example, a virus vector, a plasmid vector, an artificial chromosome vector, a transposon vector can be mentioned. As the virus vector, a retrovirus vector, an adenovirus vector, a Sendai virus vector, a lentivirus vector, an adeno-associated virus vector can be mentioned. The method of introducing these gene expression vectors into cells can be according to a known method, and is not particularly limited, but can be introduced into cells by a method such as lipofection, liposome, microinjection, etc.

[0032] In one embodiment of the present invention, it is preferable to contact pluripotent stem cells with a TGFβ inhibitor and / or a BMP inhibitor and / or a GSK3β inhibitor, and it is even more preferable to contact pluripotent stem cells with a TGFβ inhibitor, a BMP inhibitor, and a GSK3β inhibitor. The TGFβ inhibitor and / or the BMP inhibitor and / or the GSK3β inhibitor may be contacted with pluripotent stem cells before or simultaneously with the expression of the Ascl1 gene, or the Ascl1 gene and one or more genes selected from the group consisting of the Lmx1A gene, the FoxA2 gene, the Nurr1 gene, the Pitx3 gene, and the En1 gene. It is preferable to contact the pluripotent stem cells with these inhibitors before the expression of these genes. This allows dopaminergic neurons to be induced efficiently in a short period of time.

[0033] The TGFβ inhibitors that can be used in the present invention are not limited, but examples include A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide; CAS Registry Number: 909910-43-6) and SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide; CAS Registry Number: 301836-41-9). Of these, SB431542 is preferred.

[0034] BMP inhibitors that can be used in the present invention are not limited, but include dorsomorphin (CAS number: 866405-64-3), LDN-193189 (CAS number: 1062368-24-4), Noggin, and the like. Of these, dorsomorphin is preferred. In one embodiment of the present invention, the BMP inhibitor is a BMP and AMPK inhibitor that also has AMPK inhibitory activity.

[0035] GSK3 inhibitors that can be used in the present invention include, but are not limited to, CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile; CAS registration number: 252917-06-9), BIO (6-bromoindirubin-3'-oxime; CAS registration number: 667463), and the like. -62-9), Kenpaullone (9-bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one; CAS Registry Number: 142273-20-9), IM-12 (3-(4-fluorophenylethylamino)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; CAS Registry Number: 1129669-05-1), and the like. Of these, CHIR99021 is preferred.

[0036] In one embodiment of the present invention, the TGFβ inhibitor and / or BMP inhibitor and / or GSK3β inhibitor may be contacted with pluripotent stem cells by, for example, adding the inhibitor to the medium the pluripotent cells are cultured in. Furthermore, in one embodiment of the present invention, the period for contacting the pluripotent stem cells with the TGFβ inhibitor and / or BMP inhibitor and / or GSK3β inhibitor may be 1 to 10 days, preferably 2 to 8 days, more preferably 3 to 5 days, for example 4 days, before expressing the above-mentioned gene.

[0037] In the medium used in one embodiment of the present invention, the concentration of the TGFβ inhibitor and / or BMPK inhibitor and / or GSK3β inhibitor added is not particularly limited, but for example, when CHIR99021 is used as the GSK3β inhibitor, the concentration is generally 600 nmol / L to 15 μmol / L (preferably 1.5 μmol / L to 6 μmol / L); when Dorsomorphin is used as the BMP inhibitor, the concentration is generally 400 nmol / L to 10 μmol / L (preferably 1 nmol / L to 4 μmol / L); and when SB431542 is used as the TGFβ inhibitor, the concentration is generally 600 nmol / L to 15 μmol / L (preferably 1.5 μmol / L to 6 μmol / L).

[0038] In one embodiment, a cell population containing dopamine neurons produced by the production method of the present invention has a high proportion of induced dopamine neurons, and is therefore useful, for example, in disease research using dopamine neurons and in screening for new therapeutic drugs.

[0039] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0040] Various Vectors Used The following vectors were used in the examples described below. (1) Transposase expression vector: pCMV-HyPBase-PGK-Puro Ishii et al. (eNeur. 2019 Oct 17; 6(5): ENEURO.0403-18.2019) Using the custom vector construction service of VectorBuilder (VectorBuilder Japan), pCMV-HyPBase-PGK-Puro (plasmid) was designed and created (Figure 1C, upper). HyPBase is a gene encoding a transposase (piggyBac transposase) (modified to a highly active form by mutagenesis).

[0041] (2) PiggyBac vector for rtTA expression: pG-PB-CAG-rtTA3G-IH. Ishii et al. (eNeurology. 2019 Oct 17; 6(5): ENEURO. 0403-18.2019) used the custom vector construction service of VectorBuilder (VectorBuilder Japan) to design and construct the PiggyBac vector for rtTA expression, pG-PB-CAG-rtTA3G-IH (plasmid) (Figure 1C, middle). rtTA3G is a gene encoding a third-generation reverse tetracycline-controlled transactivator.

[0042] (3) PiggyBac vector for Tet-inducible hASCL1 gene expression: PB-P(tetO)-hAscl1-pA-PGK-PuroTK-pA. Based on the description of Ishii et al. (eNeurology. 2019 Oct 17;6(5):ENEURO.0403-18.2019), a PiggyBac vector for Tet-inducible hASCL1 gene expression, PB-P(tetO)-hAscl1-pA-PGK-PuroTK-pA (plasmid), was designed and constructed using the custom vector construction service of VectorBuilder (VectorBuilder Japan) (Figure 1C, middle panel). The vector contains a nucleic acid sequence encoding the full-length ORF of human ASCL1 (see SEQ ID NOs: 1 and 2).

[0043] (4) Lentiviral vector for Tet-inducible FOXA2 gene, NR4A2 gene, and EN1 gene expression: pLV-TRE3G-hFOXA2-T2A-hNR4A2-P2A-hEN1-mPGK-Bsd; and (5) Lentiviral vector for Tet-inducible LMX1A gene and PITX3 gene expression: pLV-TRE3G-hLMX1A-T2A-hPITX3-mPGK-Neo. Using the custom vector construction service of VectorBuilder (VectorBuilder Japan), we designed and constructed a plasmid (pLV-TRE3G-hFOXA2-T2A-hNR4A2-P2A-hEN1-mPGK-Bsd) for constructing a lentiviral vector that expresses the FOXA2 gene, the NR4A2 gene, and the EN1 gene in the presence of Tet, and a plasmid (pLV-TRE3G-hLMX1A-T2A-hPITX3-mPGK-Neo) for constructing a lentiviral vector that expresses the LMX1A gene and the PITX3 gene in the presence of Tet (Figure 1C, bottom). The cloning site of the plasmid contains nucleic acid sequences encoding the full-length ORFs of FOXA2 (see SEQ ID NOs: 5 and 6), NR4A2 (see SEQ ID NOs: 7 and 8), EN1 (see SEQ ID NOs: 11 and 12), LMX1A (see SEQ ID NOs: 3 and 4), and PITX3 (see SEQ ID NOs: 9 and 10).

[0044] 10 μg of the prepared plasmid was transfected into HEK293T cells and incubated at 37°C, 5% CO ２ The cells were incubated for 72 hours under an atmosphere of 0.1% CO₂. The culture supernatant was then collected, filtered through a 45 μm filter, and cryopreserved. A portion of the cryopreserved culture supernatant was used to measure the infectious titer (MOI).

[0045] Example 1 Maintenance Culture of iPS Cells The seeding density of iPS cells (414C2 strain) during subculture was 1.5 x 10 in a 6-well plate. ４The plates used for cell passage were not coated, and just before cell seeding, 1.5 mL of undifferentiated cell medium Stem Fit (AK02N, manufactured by Ajinomoto Co., Inc.) containing 10 μmol / L ROCK inhibitor Y27632 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 1.5 μg / mL iMatrix-511 (manufactured by Nippi Corporation) was poured into a 6-well plate at 1.5 mL / well, and the cells were cultured in accordance with the known cell culture method published by the Kyoto University iPS Cell Center, except that the cells were directly seeded.

[0046] Example 2: Transfection of human ASCL1 (achaete-scute family bHLH transcription factor 1) gene into iPS cells The scheme for transfection of the ASCL1 gene into iPS cells is shown in Figure 1 A. Specifically, the ASCL1 gene was transfected into iPS cells as follows.

[0047] (1) Preparation of medium and coating of plate for cell seeding after gene transfer operation Stem Fit (AK02N, Ajinomoto Co., Inc.) supplemented with 20 μmol / L Y27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) and 2.5 μg / mL iMatrix-511 (Nippi Corporation) was placed in 6 wells (1 plate) at 2 mL / well and incubated at 37°C and 5% CO ２ The mixture was incubated under an atmosphere of 0.25°C.

[0048] (2) Single cell generation of iPS cells: Approximately 1.5 x 10 cells were cultured in a 6-well plate. ４ iPS cells seeded at 100 cells / well were cultured in Stem Fit (AK02N, Ajinomoto Co., Inc.) for approximately one week. After removing the medium with an aspirator, the cells were washed with 1 mL / well of PBS(-). After removing the PBS(-), 0.5x TrypLE (registered trademark) Select [a 1:1 mixture of TrypLE Select (Thermo Fisher Scientific):PBS(-) mixed with 0.5 mol / L EDTA (pH 8.0) at a volume equivalent to 1 / 2000 of the total volume] was added at 0.5 mL / well, and the cells were incubated at 37°C, 5% CO ２After confirming that intercellular adhesion had decreased and the outlines of individual cells were somewhat visible, the cells were washed with 1 mL / well of PBS(-).

[0049] Next, the cells were rapidly detached using 1 mL of Stem Fit (AK02N, Ajinomoto Co., Inc.) containing 10 μmol / L Y27632 (Fujifilm Wako Pure Chemical Industries, Ltd.), transferred to a 15 mL or 50 mL conical tube, and thoroughly stirred. Then, 10 μL of the cell suspension was mixed with trypan blue staining solution, and the number of viable cells was counted using a hemocytometer to calculate the viable cell density of the suspension. The cell suspension was left to stand at 4°C or on ice, and a portion was cultured at 1.5 × 10 for maintenance culture. ４ The cells were seeded at 1000 cells / well onto a 6-well plate.

[0050] (3) Introduction of a vector carrying the ASCL1 gene into iPS cells. Gene Juice (#70967, Merck Millipore), a gene transfer reagent for lipofection, was warmed to room temperature and thoroughly stirred using a vortex mixer, etc. Then, 4.5 μL of Gene Juice was added to 100 μL of Opti-MEM (#31985088, Thermo Fisher Scientific) placed in a 1.5 mL tube, thoroughly stirred, and allowed to stand at room temperature for 5 minutes to prepare a lipofection reagent cocktail.

[0051] 0.5 μg each of the transposase expression vector (pCMV-HyPBase-PGK-Puro) shown in the upper panel of Figure 1C and the PiggyBac vector for rtTA expression (pG-PB-CAG-rtTA3G-IH) shown in the middle panel of Figure 1C, and 1.5 μg of the PiggyBac vector for Tet-inducible hASCL1 gene expression (PB-P(tetO)-hAscl1-pA-PGK-PuroTK-pA) were added to the lipofection reagent cocktail prepared above, stirred well, and allowed to stand at room temperature for 15 minutes to prepare a vector lipofection reagent.

[0052] While the vector lipofection reagent was left to stand for 15 minutes, 3 x 10 cells were single-celled in (2). ５The equivalent of 100 cells was transferred to a 1.5 mL tube and centrifuged at 200 G for 5 minutes. After incubation with the vector lipofection reagent, the supernatant of the centrifuged cells was removed, and the vector lipofection reagent cocktail prepared above was added to the pellet, pipetted, and allowed to stand at room temperature for 5 minutes. The obtained cells were evenly seeded at 10-20 μL / well into the cell culture plate previously incubated in (1), and incubated at 37°C, 5% CO. ２ After incubation for approximately 3 hours under an atmosphere of 0°C, the entire medium was replaced with 2 mL / well of Stem Fit (AK02N, manufactured by Ajinomoto Co., Inc.) containing 20 μmol / L Y27632 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) that had been warmed to 37°C.

[0053] (4) Selection of iPS cells that can be induced into dopaminergic neurons using antibiotics. One day after the gene transfer in (3), the entire medium was replaced (1.5 mL / well) with Stem Fit (AK02N, Ajinomoto Co., Inc.) without the addition of Y27632 or iMatrix-511, and the cells were cultured for two days. The entire medium was then replaced (2.0 mL / well) with Stem Fit (AK02N, Ajinomoto Co., Inc.) containing 10 μg / mL puromycin, and the cells were cultured for one day. The entire medium was then replaced (2.0 mL / well) with Stem Fit (AK02N, Ajinomoto Co., Inc.) supplemented with 150 μg / mL hygromycin, and the cells were cultured for one day. Thereafter, the entire medium was replaced with Stem Fit (AK02N, Ajinomoto Co., Inc.) (1.5 mL / well), and the cells were cultured for 5 days without the addition of antibiotics. The surviving iPS cells were used in Example 3, or passaged or cryopreserved as necessary.

[0054] Example 3: Introduction of the human FOXA2 (Forkhead box a2) gene, human NR4A2 (nuclear receptor subfamily 4 group A member 2) gene, human EN1 (engrailed homeobox 1) gene, human LMX1A (LIM homeobox transcription factor 1 alpha) gene, and human PITX3 (paired-like homeodomain transcription factor 3) gene into iPS cells The scheme for introducing the FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene into iPS cells is shown in Figure 1A. Specifically, the FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene were introduced into iPS cells as follows.

[0055] (1) Infection of iPS cells with lentiviral vectors for introducing FOXA2, NR4A2, EN1, LMX1A, and PITX3 genes. 1.5 × 10 iPS cells prepared by the procedures described in Example 2(1) to (4) were cultured in a 6-well plate containing Stem Fit (AK02N, Ajinomoto Co., Inc.) containing 10 μmol / L Y27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) and 1.5 μg / mL iMatrix-511 (Nippi Corporation). ４ The cells were seeded at 1000 cells / well and cultured for 1 day. Next, the entire medium was replaced with 1.5 mL / well of Stem Fit (AK02N, Ajinomoto Co., Inc.) without Y27632 or iMatrix-511. The iPS cells were then infected with a suspension of lentiviral vectors (pLV-TRE3G-hFOXA2-T2A-hNR4A2-P2A-hEN1-mPGK-Bsd) for Tet-inducible hFOXA2, NR4A2, and EN1 gene expression and a suspension of lentiviral vectors (pLV-TRE3G-hLMX1A-T2A-hPITX3-mPGK-Neo) for Tet-inducible LMX1A and PITX3 gene expression (Vector Builder) (MOI = 10.0 for each), and cultured for 1 day.

[0056] (2) Selection of lentivirus-infected iPS cells using antibiotics. One day after the addition of the lentiviral vector suspension (1), the entire medium was replaced with 1.5 mL / well of Stem Fit (AK02N, Ajinomoto Co., Inc.) without Y27632 or iMatrix-511, and the cells were cultured for one day. The entire medium was replaced with 2.0 mL / well of the aforementioned Stem Fit (AK02N, Ajinomoto Co., Inc.), and 100 μg / mL G418 (G418 Disulfate, #08973-14, Nakarai) was added and the cells were cultured for two days. After two days of culture, the entire medium was replaced with 1.5 mL / well of Stem Fit (AK02N, Ajinomoto Co., Inc.), and the cells were cultured for one day. Next, blasticidin (Blasticidin S, Hydrochloride, #KK-400, Funakoshi) was added to a final concentration of 20 μg / mL, and the cells were cultured for 1 day. After culturing for 1 day, the entire medium was replaced with Stem Fit (AK02N, Ajinomoto Co., Inc.) (1.5 mL / well), and the cells were cultured for 5 days without the addition of antibiotics. The surviving iPS cells were used in Example 4, or passaged or cryopreserved as necessary.

[0057] Example 4 Differentiation of Gene-Transfected iPS Cells into Dopamine Neurons The scheme for differentiation of iPS cells into dopamine neurons is shown in Figure 1B. Specifically, differentiation was induced as follows.

[0058] (1) Pre-coating of cell culture plates PBS(-) containing 15 μg / mL Poly-L-Ornithine solution (0.01%, #P3655, Sigma-Aldrich) was poured onto the plate and left to stand for 6 hours or more. For cell recovery tests such as RNA analysis, 6-well plates were used and coated with 1.0 mL / well of Poly-L-Ornithine solution. For tests such as immunostaining and imaging, 96-well plates were used and coated with 50 μL / well of Poly-L-Ornithine solution. Note that when coating, CO 2 for cell culture was used. ２ They were placed in an incubator to maintain a constant humidity level and prevent drying.

[0059] The day before seeding iPS cells for neural differentiation, the Poly-L-Ornithine solution was removed by suction. The 6-well plate was washed once with 2 mL / well of PBS(-), and the 96-well plate was washed once with approximately 100 μL / well of PBS(-), and the remaining liquid was thoroughly removed by suction. Next, using a 50 mL conical tube or the like, Matrigel (registered trademark) (#354277, BD Biosciences) diluted 100-fold with PBS(-) was prepared, and the 6-well plate was coated at 1.0 mL / well, and the 96-well plate was coated at 50 μL / well for 1 day. Note that during coating, CO 2 for cell culture was used. ２ They were placed in an incubator to maintain a constant humidity level and prevent drying.

[0060] (2) DSC treatment of iPS cells The iPS cells prepared in Example 3 were cultured in the same manner as in Example 1, and after 4 days of passage (Day-4), the entire medium was replaced with 1.5 mL / well of Stem Fit (AK02N, Ajinomoto Co.) containing 3 μmol / L Dorsomorphin (#P5499, Sigma-Aldrich), 3 μmol / L SB431542 (#1614, Tocris Bioscience), and 3 μmol / L CHIR99021 (#04-0004, Stemgent), and the cells were cultured for 4 days. These three compounds are collectively referred to as "DSC" below, taking the initials of each compound name (Dorsomorphin, SB43152, CHIR99021). The entire medium was replaced every day with the above-mentioned DSC-containing medium.

[0061] (3) Differentiation into dopaminergic neurons After 4 days of culture, the iPS cells for neuronal differentiation were dissociated into single cells by the same method as in (2) of Example 2. The cells were suspended in a doxycycline-containing differentiation medium containing the following mixture. Neurobasal Plus Medium (#A3582901, manufactured by Thermo Fisher Scientific, a mixture of the following as a basal medium) B27 Plus Supplement (50x, #A3582801, manufactured by Thermo Fisher Scientific) (added to the basal medium at 1 / 50) Glutamax (manufactured by Thermo Fisher Scientific) (added to the basal medium at 1 / 100) Culture one (#A3320201, manufactured by Thermo Fisher Scientific) (added to the basal medium at 1 / 100) BDNF (Recombinant human BDNF protein, #B-250, manufactured by Alomone Labs) (final concentration 20 ng / mL) GDNF (Recombinant human GDNF protein, #G-240, manufactured by Allomone Labs) (final concentration 10 ng / mL) L-Ascorbic Acid (#016-04805, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (final concentration 200 μmol / L) dbcAMP (N6,2'-O-Dibutyryladenosine-3',5'-cyclic Monophosphate Sodium Salt (#11540-61, manufactured by Nakarai) (final concentration 400 μmol / L) Doxycycline (#D4116, manufactured by Tokyo Chemical Industry Co., Ltd.) (1.0 μg / mL) Y27632 (#030-24026, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (final concentration 20 μmol / L)

[0062] The cell suspension was seeded on the plate coated with (1). 6.0 × 10 ５ cells / 3 mL / well, 2.0 × 10 cells for 96-well plates ４ The cells were evenly seeded at 200 μL / well and cultured for 5 days.

[0063] (4) Culture of dopaminergic neurons On day 5 after the start of culture, the iPS cells differentiated in (3) were subjected to half-medium exchange with a differentiation medium having the following composition: A total volume of 3.0 mL / well was poured into a 6-well plate, and a total volume of 200 μL / well was poured into a 96-well plate. Neurobasal Plus Medium (#A3582901, manufactured by Thermo Fisher Scientific, a mixture of the following as a basal medium) B27 Plus Supplement (50x, #A3582801, manufactured by Thermo Fisher Scientific) (added to the basal medium at 1 / 50) Glutamax (manufactured by Thermo Fisher Scientific) (added to the basal medium at 1 / 100) Culture one (#A3320201, manufactured by Thermo Fisher Scientific) (added to the basal medium at 1 / 100) BDNF (Recombinant human BDNF protein, #B-250, manufactured by Alomone Labs) (final concentration 20 ng / mL) GDNF (Recombinant human GDNF protein, #G-240, manufactured by Allomone Labs) (final concentration 10 ng / mL) L-Ascorbic Acid (#016-04805, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (final concentration 200 μmol / L) dbcAMP (N6,2'-O-Dibutyryladenosine-3',5'-cyclic Monophosphate Sodium Salt, #11540-61, manufactured by Nakarai (final concentration 400 μmol / L)

[0064] Thereafter, half of the medium was replaced every four days with the medium of the above composition. After 10 days from seeding in Example 4(3), the cells were collected and subjected to RNA purification, immunostaining, and the like, as described in the following Examples.

[0065] Example 5: Observation of cells 10 days after induction of expression Cells transiently expressing the ASCL1 gene of Example 2 were cultured under the above-mentioned conditions of non-DSC treatment (DSC-) or DSC treatment (DSC+). 10 days after induction of expression, cell morphology was observed using a microscope. The results are shown in Figure 2. As shown in Figure 2, DSC treatment improved cell loss associated with induction of ASCL11 expression.

[0066] Example 6: Immunostaining of cells 15 days after induction of expression Cells transiently expressing the ASCL1 gene of Example 2 were cultured under the above-mentioned conditions of non-DSC treatment (DSC-) or DSC treatment (DSC+). 15 days after induction of expression, the cells were fixed and immunostained using Anti-Tubb3 (a neuronal cell marker). Furthermore, cell nuclei were stained using Hoechst (denoted as "Ho") and observed under a fluorescent microscope. Cell immunostaining may be performed according to known methods. As shown in Figure 3, DSC treatment increased the number of neurites.

[0067] Example 7: Immunostaining of Cells 15 Days After Expression Induction Cells transiently expressing the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene, prepared in Example 3, were cultured under the above-mentioned DSC-untreated (DSC-) or DSC-treated (DSC+) conditions. On day 15 after expression induction, the cells were fixed and immunostained using anti-Tubb3 (neuron marker) antibody (Sigma-Aldrich, #T8660, mouse monoclonal antibody) and anti-TH (dopamine neuron marker) antibody (Sigma-Aldrich, #AB152, rabbit polyclonal antibody). Cell nuclei were also stained with Hoechst (denoted as "Ho") and observed under a fluorescent microscope. The results are shown in Figures 4A and 4B. As shown in Figures 4A and 4B, DSC treatment increased the Tubb3-positive rate and the TH-positive rate.

[0068] Example 8 Analysis of Peripheral Nerve Cell Marker mRNA Expression Levels by Quantitative PCR The cells transiently expressing the ASCL1 gene prepared in Example 3, and the cells transiently expressing the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene were each subjected to DSC treatment. Seven days after induction of expression, the cells were harvested, and the amount of PRPH (peripheral nerve cell marker) mRNA was analyzed by quantitative PCR. The following primers were used in quantitative PCR:

[0069] Primer sequences for human PRPH gene qPCR: Forward primer: 5'-GCCTGGAACTAGAGCGCAAG-3' (SEQ ID NO: 13) Reverse primer: 5'-CCTCGCACGTTAGACTCTGG-3' (SEQ ID NO: 14)

[0070] Human ACTB gene qPCR primer sequences (for reference): Forward primer: 5'-CACCATTGGCAATGAGCGGTTC-3' (SEQ ID NO: 15) Reverse primer: 5'-AGGTCTTTGCGGATGTCCACGT-3' (SEQ ID NO: 16)

[0071] As shown in Figure 5, compared with the group of cells transiently expressing the ASCL1 gene (denoted as "A"), the expression level of PRPH was reduced in the group of cells transiently expressing the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene (denoted as "A+5Fs").

[0072] Example 9: Transcriptome analysis using RNA-seq Cells transiently expressing the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene, as prepared in Example 3, were cultured under DSC-untreated (DSC-) and DSC-treated (DSC+) conditions. Twenty days after induction of expression, the cells were harvested and subjected to eukaryotic RNA-seq analysis using an Illumina Nova-Seq 6000. The following RNA-seq analyses were performed using the same instrument. As shown in Figure 6A, DSC treatment reduced the expression levels of cell proliferation markers (TOP2A and MKI67).

[0073] DSC treatment was performed on cells transiently expressing the ASCL1 gene, and cells transiently expressing the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene. 20 days after induction of expression, the cells were harvested and subjected to RNA-seq analysis.

[0074] As shown in Figure 6B, compared with the group of cells transiently expressing the ASCL1 gene (denoted as "A"), the expression levels of LMX1B, GBX2, and TH (dopamine neuron markers) were increased in the group of cells transiently expressing the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene (denoted as "A+5Fs").

[0075] As shown in Figure 6C, compared with the group of cells transiently expressing the ASCL1 gene (denoted "A"), the expression levels of inhibitory neuron markers DLX1 / 2 / 5 / 6, SOX6, NKX2.2, LHX6, and GAD1 were reduced in the group of cells transiently expressing the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene (denoted "A+5Fs").

[0076] Example 10: Analysis of dopamine production ability by HPLC-ECD method Doxycycline-untreated cells and cells transiently expressing the ASCL1 gene, FOXA2 gene, NR4A2 gene, EN1 gene, LMX1A gene, and PITX3 gene were cultured under DSC conditions. 21 days after induction of expression, the cells were harvested, and the amount of intracellular dopamine was measured by HPLC-ECD method.

[0077] As shown in Figure 7, dopamine neurons (referred to as "iDA") generated by this method were found to produce dopamine (referred to as "DA"), and the dopamine peripheral metabolites dihydroxyphenylacetic acid (referred to as "DOPAC") and homovanillic acid (referred to as "HVA"). In iPS(-DOX), the amounts of DA, DOPAC, and HVA were all below the detection limit.

[0078]

Claims

1. A method for inducing the differentiation of dopaminergic neurons from pluripotent stem cells, comprising: (1) contacting the pluripotent stem cells with a TGFβ inhibitor, a BMP inhibitor, and / or a GSK3β inhibitor; and (2) expressing the Ascl1 gene in the pluripotent stem cells.

2. The method according to claim 1, wherein step (1) is: (1) contacting the pluripotent stem cells with a TGFβ inhibitor, a BMP inhibitor, and a GSK3β inhibitor.

3. The method according to claim 1 or 2, wherein the TGFβ inhibitor is SB431542.

4. The method according to claim 1 or 2, wherein the BMP inhibitor is Dorsomorphin.

5. The method according to claim 1 or 2, wherein the GSK3β inhibitor is CHIR99021.

6. The method according to any one of claims 1 to 5, further comprising expressing in the pluripotent stem cells one or more genes selected from the group consisting of the Lmx1A gene, the FoxA2 gene, the Nurr1 gene, the Pitx3 gene, and the En1 gene.

7. The method according to any one of claims 1 to 6, wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells).

8. A method for producing a cell population containing dopaminergic neurons from pluripotent stem cells, comprising: (1) contacting the pluripotent stem cells with a TGFβ inhibitor, a BMP inhibitor, and / or a GSK3β inhibitor; and (2) expressing the Ascl1 gene in the pluripotent stem cells.

9. The method according to claim 8, wherein step (1) is: (1) contacting the pluripotent stem cells with a TGFβ inhibitor, a BMP inhibitor, and a GSK3β inhibitor.

10. The method according to claim 8 or 9, wherein the TGFβ inhibitor is SB431542.

11. The method according to claim 8 or 9, wherein the BMP inhibitor is Dorsomorphin.

12. The method according to claim 8 or 9, wherein the GSK3β inhibitor is CHIR99021.

13. The method according to any one of claims 8 to 12, further comprising expressing in the pluripotent stem cells one or more genes selected from the group consisting of the Lmx1A gene, the FoxA2 gene, the Nurr1 gene, the Pitx3 gene, and the En1 gene.

14. The method according to any one of claims 8 to 13, wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells).

15. A cell group containing dopamine neurons produced by the method according to any one of claims 8 to 14.

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