Transformed stem cells, nerve cells, and method for producing nerve cells derived from transformed stem cells

Abstract

The purpose of the present invention is to provide a means and a method for easily and quickly inducing the differentiation of nerve cells (for example, dopamine nerve cells or glutamic acid nerve cells) at low cost. The present invention pertains to transformed stem cells and the like in which an exogenous gene encoding each of NeuroD1, Ascl1, Brn2, Meis2, and Scrt1, which are transcription factors, or an exogenous gene encoding NeuroD1, which is a transcription factor, is introduced into stem cells.

Description

Transformed stem cells, nerve cells, and method for producing nerve cells derived from transformed stem cells

[0001] The present invention relates to transformed stem cells into which a gene encoding a specific transcription factor has been introduced, as well as to neurons derived from the transformed stem cells and methods for producing the neurons.

[0002] Conventionally, a stepwise differentiation induction method using stepwise medium replacement has been used to induce differentiation of neural cells from neural progenitor cells, pluripotent stem cells, etc. (see Non-Patent Document 1). However, such a stepwise differentiation induction method requires the preparation of a complex combination of media containing cytokines, low molecular weight compounds, etc., and also requires a long period of about one month to induce mature neural cells (e.g., dopamine neural cells (dopamine neurons)). Prior Art Literature

[0003] Hiller et al. , NPJ Regen. Med. 7:24,2022

[0004] Under these circumstances, there has been a demand for the development of means and techniques for inducing differentiation of neurons (for example, dopaminergic neurons or glutamic neurons) simply, at low cost, and quickly.

[0005] The present invention has been made in consideration of the above circumstances, and provides the following transformed stem cells, nerve cells, and methods for producing nerve cells derived from transformed stem cells.

[0006] (1) A transformed stem cell into which exogenous genes encoding the transcription factors NeuroD1, Ascl1, Brn2, Meis2, and Scrt1 have been introduced. (2) A transformed stem cell into which an exogenous gene encoding the transcription factor NeuroD1 has been introduced. (3) The transformed stem cell according to (1) or (2), wherein the stem cell is an induced pluripotent stem cell (iPS cell) or an embryonic stem cell (ES cell) derived from a mammal. (4) The transformed stem cell according to (3), wherein the mammal is a human. (5) The transformed stem cell according to (1) or (2), wherein expression of the exogenous gene is suppressed.

[0007] (6) A neuron derived from the transformed stem cell described in (1) above. (7) A neuron derived from the transformed stem cell described in (2) above. (8) A neuron according to (6) or (7) above, which is differentiated from the transformed stem cell. (9) A neuron according to (6) or (7) above, in which the transformed stem cell is in a state in which the exogenous gene is expressed. (10) A neuron according to (6) above, which is a dopamine neuron. (11) A neuron according to (10) above, which is a GABAergic neuron. Here, the neuron according to (11) is a dopamine neuron that is also a neuron capable of releasing GABA (GABAergic neuron).

[0008] (12) The neuron according to (7), which is a glutamate neuron. (13) A method for producing a neuron derived from a stem cell, comprising introducing exogenous genes encoding the transcription factors NeuroD1, Ascl1, Brn2, Meis2, and Scrt1 into a stem cell and differentiating the stem cell after the introduction. (14) A method for producing a neuron derived from a stem cell, comprising introducing an exogenous gene encoding the transcription factor NeuroD1 into a stem cell and differentiating the stem cell after the introduction. (15) The method according to (13) or (14), wherein the expression of the exogenous gene is suppressed in the stem cell upon the introduction, and the suppression of the expression of the exogenous gene is released upon differentiation. (16) The method according to (13) or (14), wherein the stem cell is a mammalian induced pluripotent stem cell (iPS cell) or embryonic stem cell (ES cell).

[0009] (17) The method according to (15), wherein the mammal is a human. (18) The method according to (13), wherein the neuronal cells are dopamine neuronal cells. (19) The method according to (18), wherein the neuronal cells are GABAergic neuronal cells. (20) The method according to (14), wherein the neuronal cells are glutamate neuronal cells. (21) A kit comprising the transformed stem cell according to (5) and an agent that derepresses the expression of the exogenous gene in the transformed stem cell. (22) The kit according to (21), which is for producing neuronal cells derived from the transformed stem cell.

[0010] (23) A differentiation induction kit comprising genes encoding the transcription factors NeuroD1, Ascl1, Brn2, Meis2, and Scrt1. (24) A differentiation induction kit comprising a gene encoding the transcription factor NeuroD1. (25) The kit according to (23), which is a kit for inducing differentiation from stem cells into dopaminergic neurons. (26) The kit according to (25), which is a kit for inducing differentiation from stem cells into GABAergic neurons. (27) The kit according to (24), which is a kit for inducing differentiation from stem cells into glutamine neurons.

[0011] According to the present invention, transformed stem cells can be provided as a means for easily, inexpensively, and rapidly inducing differentiation of neurons (e.g., dopaminergic neurons or glutamate neurons). Furthermore, neurons (e.g., dopaminergic neurons or glutamate neurons) induced to differentiate from the transformed stem cells can be provided. Furthermore, such differentiated neurons can be used for the treatment of various neurodegenerative diseases (e.g., Parkinson's disease).

[0012] Figures 1A and 1B are shown. Figure 1A shows that in this example, expression of the five introduced transcription factors (5TFs) was confirmed by RT-PCR. Figure 1B shows that in this example, differentiation into iN (induced neuron) cells expressing the neuron markers TUBB3 and Map2ab was confirmed with 100% efficiency. Figures 2A and 2B are shown. Figure 2A shows that in this example, increased gene expression of neuron marker genes TH and NR4A2, which are specifically expressed in dopaminergic neurons, and DDC, a dopa decarboxylase involved in dopamine synthesis, was confirmed. Figure 2B shows that in this example, protein expression of TH, a dopaminergic neuron marker, was confirmed by immunostaining using iN cells 14 days after induction. Figure 2C is shown. Figure 2C shows the results of single-cell RNA-seq (scRNA-seq) analysis of 5TF-iN cells in this example. Specifically, it was revealed that 5TF-iN cells express not only dopaminergic cell markers but also glutamatergic neuronal subtype cell markers, and cells co-expressing these markers were visualized by UMAP. Figure 2D shows the results of single-cell RNA-seq (scRNA-seq) analysis of 5TF-iN cells in this example. Specifically, it was revealed that 5TF-iN cells express not only dopaminergic cell markers but also GABAergic neuronal subtype cell markers, and cells co-expressing these markers were visualized by UMAP. Figure 2E shows the results of single-cell RNA-seq (scRNA-seq) analysis of 5TF-iN cells in this example. Specifically, it was revealed that 5TF-iN cells express not only dopaminergic cell markers but also GABAergic neuronal subtype cell markers, and cells co-expressing these markers were visualized by UMAP. Figure 2E shows the results of single-cell RNA-seq (scRNA-seq) analysis of 5TF-iN cells in this example. Specifically, it was revealed that 5TF-iN cells also express GABAergic and glutamatergic neuronal subtype cell markers, and cells co-expressing these markers were visualized by UMAP. Figures 3A, 3B, and 3C are shown. Figure 3A shows that in this example, differentiation-induced neurons (5TF-iN cells) showed a dramatic Ca upregulation immediately after KCL stimulation.２＋ 3B shows that in this example, KCl stimulation of 5TF-iN cells increased the amount of dopamine released into the culture medium. FIG. 3C shows that in this example, KCl stimulation of 5TF-iN cells increased the amount of GABA released into the culture medium. FIGS. 4A and 4B are shown. FIG. 4A shows that in this example, differentiation-induced neurons (ND1-iN cells) barely expressed NR4A2, a dopamine neuron marker, but highly expressed VGLUT1, a glutamate receptor marker (glutamate neuron marker), confirming that ND1-iN cells are not dopamine neurons but a different subtype of neurons from 5TF-iN cells. FIG. 4B shows that immunostaining confirmed that NR4A2 was not expressed in ND1-iN cells.

[0013] The present invention will be described in detail below. The scope of the present invention is not limited to these descriptions, and other than the following examples, modifications may be made as appropriate without departing from the spirit of the present invention. This specification encompasses the entire specification of Japanese Patent Application No. 2023-045232 (filed March 22, 2023), which is the basis for claiming priority of this application. In addition, all publications cited in this specification, such as prior art documents, published patent applications, patent publications, and other patent documents, are incorporated herein by reference in their entirety, regardless of their purpose.

[0014] The present invention will be described in detail below. The scope of the present invention is not limited to these descriptions, and other than the following examples, appropriate modifications can be made without departing from the spirit of the present invention. This specification encompasses the entire specification of Japanese Patent Application No. 2024-093278 (filed June 7, 2024), from which priority is claimed. All publications cited in this specification, such as prior art documents, published patent applications, patent publications, and other patent documents, are incorporated herein by reference.

[0015] 1. Transformed Stem Cells The present invention provides transformed stem cells into which an exogenous gene encoding a specific transcription factor has been introduced.

[0016] In the present invention, specific transcription factors include (i) a combination of NeuroD1, Ascl1, Brn2, Meis2, and Scrt1, or (ii) NeuroD1. Registered information on gene sequences encoding each of the above transcription factors is, for example, as follows:

[0017] NeuroD1 (Neurogenic differentiation 1): GenBank accession number: NM_010894.3 Ascl1 (Achaete-scute homolog 1): GenBank accession number: NM_008553.5 Brn2 (Brain-specific homeobox / POU domain protein 2): GenBank accession number: NM_00889.2 Meis2 (Meis homeobox 2): GenBank accession number: NM_001136072.2 Scrt1 (Scratch family transcriptional repressor 1): GenBank accession number: NM_130893.3

[0018] Techniques for introducing and expressing an exogenous gene encoding the above-mentioned specific transcription factor into stem cells include, but are not limited to, transfecting cells with mRNA of the transcription factor, inducing expression using an RNA virus such as a Sendai virus vector, introducing a gene construct that induces expression using a DNA virus such as a lentivirus vector, and introducing a gene construct that induces expression using genetic modification such as genome editing. In particular, mRNA transfection or expression induction using an RNA virus is preferred, as there is no risk of the exogenous (foreign) gene being incorporated into the genome and remaining there for a long period of time. The present inventors have discovered that expressing an exogenous gene encoding the above-mentioned specific transcription factor in transformed stem cells can cause the cells to differentiate (induce differentiation) into neurons with specific properties.

[0019] As for the nerve cells having the specific properties, when the combination of transcription factors (i) (i.e., NeuroD1, Ascl1, Brn2, Meis2, and Scrt1) is expressed, they can be differentiated (inducible to differentiate) into dopamine nerve cells (dopamine neurons; also called dopaminergic neurons), and when the transcription factor (ii) (i.e., NeuroD1) is expressed, they can be differentiated (inducible to differentiate) into glutamatergic nerve cells (glutamatergic neurons; also called glutamatergic neurons). Here, dopaminergic neurons can generally be explained as neurons that secrete dopamine, a neurotransmitter, and play an important role in neural functions such as voluntary movement, emotion, addiction, and stress. Furthermore, glutamatergic neurons can generally be explained as neurons that are responsible for fast excitatory transmission in the central nervous system and function mainly as projection neurons throughout a wide area of ​​the brain. In the present invention, the neurons having the specific properties, i.e., dopamine neurons (dopamine neurons) and glutamate neurons (glutamate neurons), may be neurons (so-called multi-transmitter neurons) that have the function of expressing (releasing) not only dopamine and glutamate but also other neurotransmitters in a multiplicity. For example, the dopamine neurons (dopamine neurons) that are induced to differentiate when the transcription factors of the combination (i) are expressed are not particularly limited, but preferably include those that also express (release) GABA (Gamma-Amino Butyric Acid). In other words, the dopamine neurons of the present invention preferably also have the function of GABA neurons (GABA neurons; also called GABAergic neurons). Here, GABAergic neurons can generally be described as nerve cells that use the inhibitory neurotransmitter GABA as their main neurotransmitter, and that play an important role in regulating neurotransmission at synapses and maintaining normal brain activity (suppressing neural activity in the brain and suppressing excitation).

[0020] Here, the present invention can also provide an invention relating to a differentiation induction kit comprising genes encoding the transcription factors NeuroD1, Ascl1, Brn2, Meis2, and Scrt1. The kit is preferably a kit for inducing differentiation from stem cells into dopaminergic neurons. The present invention can also provide an invention relating to a differentiation induction kit comprising a gene encoding the transcription factor NeuroD1. The kit is preferably a kit for inducing differentiation from stem cells into glutamine neurons. In each of the above kits, the means and form for stably storing or maintaining the genes encoding each transcription factor may be any means and form capable of stably storing or maintaining nucleic acids. Note that preferred examples of the use of each of the above kits, specifically the method of introducing and expressing a gene encoding each transcription factor into cells to be induced to differentiate (e.g., stem cells), include a method of transfecting cells with mRNA of the transcription factor, a method of inducing expression using an RNA virus such as a Sendai virus vector, a method of introducing a gene construct that induces expression using a DNA virus such as a lentivirus vector, and a method of introducing a gene construct that induces expression using gene modification such as genome editing. Among these, the method of transfecting mRNA or inducing expression using an RNA virus is preferred, as it does not involve the risk of an exogenous (foreign) gene being incorporated into the genome and remaining there for a long period of time.

[0021] The transformed stem cells of the present invention are preferably, but not limited to, those in which the expression of an exogenous gene encoding the specific transcription factor described above (the exogenous gene after being introduced into the stem cell) is suppressed. By releasing this suppressed state at the desired timing (i.e., by expressing the exogenous gene), the transformed stem cells can be differentiated (inducing differentiation) into neurons at the desired timing. The method for suppressing the expression of an exogenous gene in transformed stem cells is not limited, and various known gene expression control methods can be employed, such as the Tet-on system. Generally, the Tet-on / off system is a method for reversibly regulating the expression of a target gene in cells or an individual animal by administering doxycycline (Dox), a derivative of the antibiotic tetracycline. This technique utilizes the Tet repressor (TetR) and Tet operator sequence (tetO sequence) that function in the E. coli tetracycline resistance operon, taking advantage of the property that TetR binds to the tetO sequence in the absence of tetracycline but is no longer able to bind to the tetO sequence when tetracycline is bound. A system that does not express a target gene (exogenous gene in this invention) in the absence of Dox but expresses the target gene in the presence of Dox is called the Tet-On system.

[0022] The present invention also provides a kit comprising transformed stem cells in which the expression of an exogenous gene encoding a specific transcription factor (the exogenous gene after introduction into the stem cell) is suppressed, as described above, and an agent that de-suppresses the expression of the exogenous gene in the transformed stem cell. The kit is preferably a kit for producing neurons (preferably, dopaminergic neurons or glutamateergic neurons) derived from the transformed stem cell. The agent that de-suppresses the expression of the exogenous gene is not limited, but preferred examples include the aforementioned doxycycline (Dox). In the kit, the means and form for stably storing or maintaining the transformed stem cells may be any means and form that allows for stable storage or maintenance of stem cells.

[0023] In the present invention, preferred stem cells into which exogenous genes encoding the specific transcription factors described above are introduced are, for example, pluripotent stem cells. Pluripotent stem cells refer to undifferentiated cells that possess both the "self-renewal ability" that allows proliferation while maintaining an undifferentiated state and the "pluripotency" that allows differentiation into all or some of the three germ layer lineages, particularly neural cells. Examples of pluripotent stem cells include embryonic stem cells (ES cells), primordial germ stem cells (EG cells), pluripotent germline stem cells (mGS cells), neural stem cells isolated from adult or fetal brains, somatic stem cells such as mesenchymal stem cells and stromal cells isolated from bone marrow, blood, or adult tissues, and induced pluripotent stem cells (iPS cells). In the present invention, preferred pluripotent stem cells are induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).

[0024] In the present invention, stem cells (preferably pluripotent stem cells) are preferably derived from mammals. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as humans, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees. Humans are particularly preferred. Furthermore, stem cells (preferably pluripotent stem cells) may be wild-type cells, wild-type cells modified with desired genes, or cells derived from patients with neurological disorders (e.g., Parkinson's disease). Embryonic stem cells (ES cells) are available from designated institutions and are also commercially available. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University.

[0025] Human and mouse induced pluripotent stem cells (iPS cells) can be established according to WO 2004 / 092357 and WO 2007 / 069666. Alternatively, a method for producing iPS cells using three factors (representative examples of which are Oct3 / 4, Klf4, and Sox2) excluding the c-Myc gene (Takahashi, K. and Yamanaka, S., Cell, 126:663-676 (2006)) or a method for inducing iPS cells using a plasmid or episomal vector without incorporating reprogramming factors into the genome (Okita, K. et al., Science, 322:949-953 (2008); Yu, J. et al., Science, 324:797-801 (2009)) may also be employed. iPS cells can be established from somatic cells such as skin fibroblasts according to the methods described in the above-mentioned documents. iPS cells can be obtained from a designated institution; for example, the 201B7 strain of human iPS cells is available from the RIKEN BioResource Center. iPS cells can be cultured and maintained, for example, according to the protocol provided by the Center for iPS Cell Research and Application, CiRA, Kyoto University (chrome-extension: / / efaidnbmnnnibpcajpcglclefindmkaj / https: / / www.cira.kyoto-u.ac.jp / j / research / img / protocol / hipsprotocolFf_140311.pdf), as described in the Examples below (for example, StemFit AK02N (Ajinomoto) can be used as the culture medium).

[0026] The present invention may also provide a method for producing the above-mentioned transformed stem cells, i.e., transformed stem cells into which an exogenous gene encoding a specific transcription factor has been introduced. Specifically, a method for producing transformed stem cells may be provided, which comprises introducing an exogenous gene encoding the specific transcription factor into stem cells, preferably such that the expression of the exogenous gene is suppressed (however, such that the suppression can be released at a desired time by an expression control system such as the Tet-on system).

[0027] 2. Neuronal Cells The present invention provides neuronal cells derived from the transformed stem cells of the present invention, and preferably provides neuronal cells differentiated (induced to differentiate) from the transformed stem cells of the present invention.

[0028] The transformed stem cells of the present invention that can be differentiated (induced to differentiate) into neurons are those in a state in which an exogenous gene encoding the specific transcription factor described above is expressed intracellularly. For example, if the transformed stem cells of the present invention have the expression of the exogenous gene suppressed by an expression control system such as the Tet-on system, this state can be exemplified by a state in which the suppression is released using the system (in the presence of Dox).

[0029] The differentiated neurons become dopaminergic neurons when the aforementioned (i) combination of transcription factors NeuroD1, Ascl1, Brn2, Meis2, and Scrt1 is expressed, and become glutamic neuronal cells when the aforementioned (ii) transcription factor NeuroD1 is expressed. The explanation of dopaminergic neurons and glutamic neuronal cells is as described in Section 1 above.

[0030] Based on the above explanation, the present invention also provides the following method for producing neurons (hereinafter also referred to as the production method of the present invention). Specifically, a method for producing stem cell-derived neurons may be provided, comprising introducing (i) exogenous genes encoding the transcription factors NeuroD1, Ascl1, Brn2, Meis2, and Scrt1, or (ii) an exogenous gene encoding the transcription factor NeuroD1, into stem cells and differentiating the stem cells (transformed stem cells) after the introduction. In this production method, for example, it is preferable that when the exogenous gene is introduced into the stem cells, expression of the exogenous gene is suppressed in the stem cells, and when the stem cells are differentiated after the introduction, the suppression of expression of the exogenous gene is released. As described above, a known gene expression control system, such as the Tet-on system, can be used to achieve the suppression and release of the suppression.

[0031] Media that can be used in the production method of the present invention include, for example, StemFit AK02N for culturing and maintaining transformed stem cells, and iN medium for obtaining neurons by differentiation.

[0032] The medium that can be used may be a serum-containing medium or a serum-free medium, but a serum-free medium is preferred. The term "serum-free medium" refers to a medium that does not contain unconditioned or unpurified serum, but also includes media containing purified blood-derived components or animal tissue-derived components (e.g., growth factors). Examples of serum-free media include iN medium.

[0033] The medium that can be used in the production method of the present invention may contain other components, such as amino acids, pyruvic acid, 2-mercaptoethanol, cytokines, antibiotics, and growth factors, at appropriate concentrations, as needed.

[0034] The culture vessel used in the production method of the present invention is not particularly limited as long as it is a known cell culture vessel, and can be appropriately selected from, for example, a flask, a tissue culture flask, a dish, a Petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a multi-plate, a multi-well plate, a chamber slide, a Petri dish, a tube, a tray, a culture bag, and a roller bottle.

[0035] In the production method of the present invention, differentiation (differentiation induction) into nerve cells is carried out in vitro. For example, typical culture conditions include a culture temperature of 32 to 40°C, a CO ２ The concentration is 2 to 10%, and the culture period is 3 to 30 days, preferably 5 to 20 days, and more preferably 7 to 14 days.

[0036] In the production method of the present invention, a substance known as a differentiation inducer into nerve cells (or nervous system cells) may be used in combination at an appropriate concentration, such as NGF, BDNF, NT3, retinoic acid, FGF, BMP inhibitory factor, IGF, GDNF, and CNTF.

[0037] The neurons of the present invention (including neurons obtained by the production method of the present invention) can be used for the treatment or prevention of various nervous system diseases and neurodegenerative diseases, including, but not limited to, Parkinson's disease and Alzheimer's disease.

[0038] Therefore, the present invention may also include inventions relating to, for example: a pharmaceutical composition for treating or preventing nervous system diseases and neurodegenerative diseases, comprising the neurons of the present invention (including neurons obtainable by the production method of the present invention); a therapeutic or preventive agent for nervous system diseases and neurodegenerative diseases, comprising the neurons of the present invention (including neurons obtainable by the production method of the present invention); a method for treating or preventing nervous system diseases and neurodegenerative diseases, comprising administering to a subject the neurons of the present invention (including neurons obtainable by the production method of the present invention) or the pharmaceutical composition; the neurons of the present invention (including neurons obtainable by the production method of the present invention) for use in the treatment or prevention of nervous system diseases and neurodegenerative diseases; use of the neurons of the present invention (including neurons obtainable by the production method of the present invention) for the manufacture of a medicament for the treatment or prevention of nervous system diseases and neurodegenerative diseases; and use of the neurons of the present invention (including neurons obtainable by the production method of the present invention) for the treatment or prevention of nervous system diseases and neurodegenerative diseases.

[0039] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples.

[0040] 1. Materials and Methods (i) iPS Cell Culture iPS cells were cultured and maintained according to the protocol provided by the Center for iPS Cell Research and Application (CiRA), Kyoto University (chrome-extension: / / efaidnbmnnnibpcajpcglclefindmkaj / https: / / www.cira.kyoto-u.ac.jp / j / research / img / protocol / hipsprotocolFf_140311.pdf). StemFit AK02N (Ajinomoto) medium was used for the culture and maintenance.

[0041] (ii) Virus Production. Lentivirus was produced by transfecting HEK293T cells in 10-cm dishes with pCMV-VSV-G-RSV-Rev and pCAG-HIVgp using polyethyleneimine. Because lot-to-lot variation in the FBS preparation added to the culture medium significantly affects the tropism of the resulting virus, FBS was avoided in virus preparation. After transfection, the cells were cultured for 2 days in 5 mL of serum-free N2 medium (DMEM / F12 supplemented with insulin (25 μg / mL), apotransferrin (100 μg / mL), progesterone (20 nM), putrescine (60 μM), and sodium selenite (30 nM)). The supernatant was collected and filtered through a 0.22 μm filter to remove cellular debris before use in virus infection experiments.

[0042] (iii) Creation and Isolation of iPS Cell Clones In this example, a lentiviral vector controlling gene expression under the tetracycline operator was used to induce neural cells. 2x10^5 iPS cells were seeded onto a 3.5 cm dish, and after confirming that the cells had adhered to the bottom of the dish, the iPS cells were infected with lentivirus containing five types of DOX-inducible transcription factors (NeuroD1 (hereinafter also referred to as ND1), Ascl1, Brn2, Meis2, and Scrt1) (5 transcription factors: 5TFs), as well as a reverse tetracycline transactivator (FUW-M2rtTA) and GFP (FUW-GFP) as an indicator of gene transfer, for gene transfer. The following day, the medium was replaced with Stemfit medium to remove the viral solution, and the cells were cultured for two days. After two days, the iPS cells were detached from the dish, and the cell suspension was subjected to limiting dilution so that 0.5 cells were placed in each well of a 96-well plate. Two hours after seeding, it was confirmed that only one cell was present in each well; wells containing multiple cells were not used in the experiment. The iPS colonies that had grown from a single cell were observed under a fluorescence microscope to determine whether GFP fluorescence was present. Only wells that showed fluorescence were retained and continued to be cultured to establish iPS clone cells.

[0043] (iv) Confirmation of gene transfer by RT-PCR RT-PCR was used to examine whether the five transgenic transcription factors were transferred into the established iPS clone cells. The iPS clone cells were treated with doxycycline (1 μg / ml) to activate the expression of the transgenes. After two days, RNA was extracted from the cells, cDNA was synthesized, and PCR was performed. The RT-PCR primers used were designed to detect only the exogenous gene expression of each transcription factor. The same method was also used to confirm the transfer of iPS cell clones expressing only ND1. The same method was also used to obtain iPS clone cells transfected with only ND1.

[0044] (v) Induction of iN (induced neuron) cells. The medium for iPS cells seeded on the dish was replaced with iN medium (Neurobasal Medium (GIBCO), 2% B27 (GIBCO), 1% Glutamax (GIBCO), penicillin / streptomycin / fungizone (HyClone), BDNF, GDNF, and NT3 (10 ng / ml each, Peprotech)), and doxycycline (1 μg / ml) was added once to activate the expression of the transgene. Half of the medium was replaced every 2-3 days during the culture period.

[0045] (vi) Immunostaining Immunostaining was performed using cells 1 or 2 weeks after Dox administration. Cells were fixed with 4% paraformaldehyde for 10 minutes. Then, cells were blocked with blocking solution (5% FBS, 0.3% Triton X-100) for 30 minutes and incubated at room temperature for 2 hours with the following primary antibodies: anti-GFAP (1:500), anti-TUBB3 (1:500), anti-TH (1:500), anti-NeuN (1:500), anti-NR4A2 (1:500), anti-MAP2 (1:500), and anti-Map2ab (1:500). Stained cells were visualized and images were acquired using a confocal laser scanning microscope (LSM800, Zeiss).

[0046] (vii) Ca ２＋ Functional analysis by imaging Ca ２＋Imaging was performed 2 weeks after Dox administration. For calcium imaging, cells were washed with recording medium (10 mM HEPES, 140 mM NaCl, 5.0 mM KCl, 1.2 mM MgCl, 2 mM CaCl, 10 mM glucose) and then incubated with recording medium containing 2 μM Fluo4-AM (Invitrogen) and 0.01% Cremophor-EL (Sigma) for 20 min at room temperature. The medium was then replaced with fresh recording medium, and 60 mM KCL was added to stimulate the cultured neurons. Calcium signals were recorded using a confocal microscope (LSM800, Zeiss).

[0047] (viii) Isolation of mRNA and cDNA Library Preparation mRNA was isolated from 5TF iPS-iN cells on day 14 after doxycycline treatment using Dynabeads mRNA DIRECT Micro Kit (Invitrogen), and a cDNA library was constructed from the purified mRNA using NEB Next Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) according to the attached protocol. The cDNA library was purified using AMPure XP beads (Beckman Coulter), and the quality of the cDNA library was assessed using a High Sensitivity D1000 Screen Tape assay (Agilent).

[0048] (ix) RNA-seq Analysis RNA-seq was performed using 150 bp pair-end sequencing on an Illumina Novaseq X plus. The resulting reads were trimmed to remove short reads (<30 bp) and low-quality reads using the FASTX toolkit (Patel and Jain, PLoS ONE 7, e30619 (2012)). The processed reads were aligned to the human reference genome hg38 using TopHat (Kim et al., Genome Biol. 14, R36 (2013)). Read counting was performed using Feature Counts software (Liao et al., Bioinformatics 30, 923-930 (2014)).

[0049] (x) Measurement of dopamine levels Dopamine levels were quantified using a Dopamine ELISA Kit (Abnova). The measurement method followed the attached protocol. Cell culture medium was used for the measurement, and the culture medium was culture medium 4 weeks after Dox administration. After treatment with KCL for 15 minutes, the culture medium was collected and used.

[0050] (xi) Measurement of GABA Amount GABA amount was measured using a GABA ELISA Kit (Abnova). The measurement method followed the attached protocol. Cell culture medium was used for the measurement, and the culture medium was culture medium 4 weeks after Dox administration. KCL was treated for 15 minutes, and then the culture medium was collected and used.

[0051] (xii) Preparation of libraries for single cell RNA-seq (scRNA-seq) analysis. 5TF iPS-iN cells 14 days after doxycycline treatment were detached with trypsin, and 10,000 cells were collected. A cDNA library was constructed using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) according to the attached protocol. Library quality was assessed using a High Sensitivity D1000 Screen Tape assay (Agilent).

[0052] (xiii) scRNA-seq analysis. scRNA-seq was performed using 150-bp pair-end sequencing on an Illumina Novaseq X Plus. The resulting Fastq files were demultiplexed before being input into Cell Ranger (v2.2.0), where each read was mapped to the human reference genome hg38 and assigned to a single cell. The data were visualized using the RunUMAP function, a nonlinear dimensionality reduction algorithm in Seurat.

[0053] 2. Experimental Results (i) Generation of 5TF-iPSC clone cells and their induction into iN cells. Five transcription factors, NeuroD1 (ND1), Ascl1, Brn2, Meis2, and Scrt1, were transfected into iPS cells (5TF (transcription factor)-iPSCs) using lentivirus, and single clones were obtained from these cells. The expression of the five introduced transcription factors was confirmed by RT-PCR in the obtained iPSC clone cells (Figure 1A). The obtained iPSC clone cells were treated with doxycycline and fixed with paraformaldehyde after 7 days. The induction of iN (induced neuron) cells was confirmed by immunostaining. As a result, we confirmed that iPS cells differentiated into iN cells expressing the neuronal markers TUBB3 and Map2ab with 100% efficiency (Figure 1B). Next-generation analysis (RNA-seq) was performed on the resulting iPS clone cells to determine the subtypes of neurons differentiated by 5TF introduction. We confirmed increased gene expression of the neuronal marker genes TH and NR4A2, which are specifically expressed in dopaminergic neurons, and DDC, a dopaminergic decarboxylase involved in dopamine synthesis (Figure 2A). Furthermore, we confirmed the protein expression of the dopaminergic neuronal marker TH by immunostaining using iN cells 14 days after induction (Figure 2B). These results suggest that iN cells induced by 5TF are dopaminergic neurons.

[0054] For more detailed analysis, we performed scRNA-seq analysis of 5TF-iN cells. Results revealed that 5TF-iN cells express not only dopaminergic cell markers but also markers for GABAergic and glutamatergic neuronal subtypes. Figures 2C–E show UMAP visualization of cells co-expressing each subtype neuronal marker. Each dot represents a single cell, and black dots (dots with a darker density than the surrounding area) indicate co-expression of two distinct subtype markers. We found cells co-expressing KCNJ6, a known dopaminergic cell marker, and SLC17A6, a glutamatergic marker (Figure 2C), cells co-expressing KCNJ6 and GAD1, a GABAergic neuronal marker (Figure 2D), and cells co-expressing GAD1 and SLC17A6 (Figure 2E).

[0055] (ii) Functional analysis of 5TF-iN cells Next, we investigated whether 5TF-iN cells function as neurons, and further whether they function as dopaminergic neurons. ２＋ The indicator Fluo4-AM was taken up into 5TF-iN cells on day 14 of induction to measure intracellular Ca ２＋ After making the concentration dynamic changes visible, depolarization stimulation was given by treatment with high concentrations of KCL. As a result, a dramatic increase in Ca ２＋It was observed that the concentration of dopamine in the 5TF-iN cells increased (Figure 3A). These results demonstrated that the 5TF-iN cells were functional neurons capable of responding to delocalization stimulation with high KCl concentrations. Furthermore, to investigate whether the 5TF-iN cells functioned as dopaminergic neurons, the amount of dopamine released into the culture medium was quantified. For this experiment, 5TF-iN cell culture medium at 4 weeks of induction was used. Measurement of the amount of dopamine in the culture medium confirmed that dopamine was released into the medium. Furthermore, it was confirmed that the amount of dopamine released into the culture medium increased with KCl stimulation (Figure 3B). These results demonstrated that the 5TF-iN cells were functional dopaminergic neurons. Furthermore, because scRNA-seq analysis showed that 5TF-iN cells expressed markers of not only dopaminergic neurons but also GABAergic neurons, the amount of GABA released into the culture medium was quantified. In this example, 5TF-iN cell culture medium at 4 weeks of induction was used. Measurement of the amount of GABA in the culture medium confirmed that GABA was released into the medium. Furthermore, it was confirmed that the amount of GABA released into the culture medium increased with KCl stimulation (Figure 3C). These results demonstrate that 5TF-iN cells release not only dopamine but also GABA.

[0056] (iii) Creation of ND1-iPSC clones and their induction into iN cells. Using the same method as used to create 5TF-iPSC clones, cloned iPSCs were generated by expressing only ND1. To examine the differences between iN cells derived from the ND1-iPSC clones and 5TF-iN cells, next-generation analysis was performed using ND1-iN cells 14 days after induction. As a result, unlike 5TF-iN cells, ND1-iN cells showed little expression of the dopaminergic neuron marker NR4A2 (Figure 4A). Meanwhile, expression of the glutamate receptor marker VGLUT1 was barely observed in 5TF-iN cells, but was highly expressed in ND1-iN cells. This revealed that ND1-iN cells are not dopaminergic neurons, but represent a different subtype of neurons from 5TF-iN cells, which highly express glutamate neuron markers (Figure 4A). Immunostaining was also performed to confirm the expression of NR4A2, and similar to the gene expression pattern, it was confirmed that it was not expressed in ND1-iN cells (Figure 4B). Based on these findings, it is highly likely that ND1-iN cells are neurons that can function as glutamate neurons.

[0057] According to the present invention, transformed stem cells can be provided as a means for easily, inexpensively, and rapidly inducing differentiation of neurons (e.g., dopaminergic neurons or glutamate neurons). Furthermore, neurons (e.g., dopaminergic neurons or glutamate neurons) induced to differentiate from the transformed stem cells can be provided. Furthermore, such differentiated neurons can be used for the treatment of various neurodegenerative diseases (e.g., Parkinson's disease).

Claims

1. Transformed stem cells into which exogenous genes encoding the transcription factors NeuroD1, Ascl1, Brn2, Meis2 and Scrt1 have been introduced.

2. Transformed stem cells in which an exogenous gene encoding the transcription factor NeuroD1 has been introduced into the stem cells.

3. The transformed stem cell according to claim 1 or 2, wherein the stem cell is an induced pluripotent stem cell (iPS cell) or an embryonic stem cell (ES cell) derived from a mammal.

4. The transformed stem cell of claim 3, wherein the mammal is a human.

5. A transformed stem cell according to claim 1 or 2, in which expression of the exogenous gene is suppressed.

6. A neuron derived from the transformed stem cell of claim 1.

7. A neuron derived from the transformed stem cell described in claim 2.

8. A nerve cell according to claim 6 or 7, which is differentiated from the transformed stem cell.

9. A neuron according to claim 6 or 7, wherein the transformed stem cell is in a state in which the exogenous gene is expressed.

10. The neuron according to claim 6, which is a dopamine neuron.

11. The neuron according to claim 10, which is a GABAergic neuron.

12. The neuron according to claim 7, which is a glutamate neuron.

13. A method for producing nerve cells derived from stem cells, comprising introducing exogenous genes encoding the transcription factors NeuroD1, Ascl1, Brn2, Meis2, and Scrt1 into stem cells and differentiating the stem cells after the introduction.

14. A method for producing nerve cells derived from stem cells, comprising introducing an exogenous gene encoding the transcription factor NeuroD1 into stem cells and differentiating the stem cells after the introduction.

15. A method according to claim 13 or 14, wherein the exogenous gene is suppressed in expression in the stem cells when introduced, and the suppression of expression of the exogenous gene is released when the stem cells are differentiated.

16. The method according to claim 13 or 14, wherein the stem cells are induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells) derived from a mammal.

17. The method of claim 15, wherein the mammal is a human.

18. The method of claim 13, wherein the neuronal cells are dopamine neuronal cells.

19. The method of claim 18, wherein the neuron is a GABA neuron.

20. The method of claim 14, wherein the neuronal cells are glutamate neuronal cells.

21. A kit comprising the transformed stem cell according to claim 5 and an agent that derepresses the expression of the exogenous gene in the transformed stem cell.

22. The kit according to claim 21, which is for producing nerve cells derived from the transformed stem cells.

23. A differentiation induction kit comprising genes encoding the transcription factors NeuroD1, Ascl1, Brn2, Meis2, and Scrt1.

24. A differentiation induction kit containing a gene encoding the transcription factor NeuroD1.

25. The kit according to claim 23, which is a kit for inducing differentiation of stem cells into dopaminergic neurons.

26. The kit according to claim 25, which is a kit for inducing differentiation of stem cells into GABAergic neurons.

27. The kit according to claim 24, which is a kit for inducing differentiation of stem cells into glutamine neurons.

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