A method for producing astrocyte-like cells

Abstract

This method for producing astrocyte-like cells includes: a step (A) for performing the upregulation of transcription factors including SPY-box transcription factor 9 (SOX9), nuclear factor IA (NFIA) and nuclear factor IB (NFIB) in human pluripotent stem cells; and a step (B) for culturing the human pluripotent stem cells in which upregulation of transcription factors has been performed, and, consequently, differentiating the human pluripotent stem cells into astrocyte-like cells.

Description

[Technical Field]

[0001] The present invention relates to a method for producing astrocyte-like cells. More specifically, the present invention relates to a method for producing astrocyte-like cells, astrocyte-like cells, a co-culture of astrocyte-like cells and neuron-like cells, and use of a medium for producing astrocyte-like cells. This application claims priority based on Japanese Patent Application No. 2020-152180, filed on September 10, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] There is a demand for the use of nervous system tissues for basic research and elucidation of nervous system diseases. However, experimental results obtained using nervous system tissues from laboratory animals such as mice and rats can be difficult to extrapolate to humans, and there are limitations to the use of human nervous system tissues. For this reason, the generation and use of human nervous system tissues in vitro has been investigated.

[0003] However, brain organoids created from pluripotent stem cells such as induced pluripotent stem (iPS) cells are known to be composed mainly of neurons and neural stem cells, and do not contain fully mature glial cells (astrocytes, oligodendrocytes, microglia, etc.).

[0004] Glial cells are responsible for the survival and functional expression of neurons, such as by providing nutrients to neurons, and it has become clear that glial cells, as well as neurons, play an important role in the expression of brain function. For this reason, there is a demand for technology to produce glial cells in vitro.

[0005] For example, Non-Patent Document 1 describes that the SRY-Box Transcription Factor 9 (SOX9) gene and the nuclear factor IB (NFIB) gene were overexpressed in human pluripotent stem cells, resulting in the induction of differentiation into astrocyte-like cells.

[0006] Furthermore, Non-Patent Document 2 describes that the nuclear factor IA (NFIA) gene, or the SOX9 gene and the NFIA gene, were overexpressed in human pluripotent stem cells, thereby inducing differentiation into astrocyte-like cells. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Canals I., et al., Rapid and efficient induction of functional astrocytes from human pluripotent stem cells, Nat Methods., 693-696, 2018. [Non-patent document 2] Li X., et al., Fast Generation of Functional Subtype Astrocytes from Human Pluripotent Stem Cells, Stem Cell Reports, 998-1008, 2018. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a technique for efficiently producing astrocyte-like cells in vitro. [Means for solving the problem]

[0009] The present invention includes the following aspects. [1] A method for producing astrocyte-like cells, comprising: (A) upregulating transcription factors, including SRY-Box Transcription Factor 9 (SOX9), nuclear factor IA (NFIA), and nuclear factor IB (NFIB), in human pluripotent stem cells; and (B) culturing the human pluripotent stem cells in which the transcription factors are upregulated, thereby differentiating the human pluripotent stem cells into astrocyte-like cells. [2] The method for producing astrocyte-like cells according to [1], wherein in step (B), the human pluripotent stem cells are cultured in a pluripotent stem cell culture medium or a neuronal culture medium. [3] The method for producing astrocyte-like cells according to [1] or [2], wherein step (A) is carried out by introducing a vector containing a nucleic acid encoding the transcription factor into the human pluripotent stem cells. [4] A method for producing astrocyte-like cells according to [1] or [2], wherein step (A) is carried out by adding or removing a tetracycline antibiotic to the culture medium of the human pluripotent stem cells into which a vector containing a nucleic acid encoding the transcription factor under the control of a tetracycline response factor has been introduced. [5] The method for producing astrocyte-like cells according to [3] or [4], wherein the vector contains a nucleic acid having a base sequence represented by the following formula (1): F 1 -S 1 -F 2 -S 2 -F 3 …(1) [In formula (1), F 1 , F 2 and F 3 represents, in no particular order, a nucleotide sequence encoding SOX9, a nucleotide sequence encoding NFIA, and a nucleotide sequence encoding NFIB; S 1 and S 2 represents the separator base sequence.] [6] The method for producing astrocyte-like cells according to any one of [3] to [5], wherein the vector is a transposon vector. [7] The method for producing astrocyte-like cells according to any one of [1] to [6], wherein in step (B), the abundance of SOX9, NFIA and NFIB in the human pluripotent stem cells is 1:1:1 in molar ratio. [8] The method for producing astrocyte-like cells according to any one of [1] to [7], wherein the human pluripotent stem cells are human induced pluripotent stem cells. [9] Astrocyte-like cells produced by the production method described in any one of [1] to [8].

[10] Co-culture of astrocyte-like cells and neuron-like cells as described in [9].

[11] Use of a medium containing basic fibroblast growth factor or a medium containing at least one factor selected from the group consisting of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, neurotrophin 3, and dibutyryl cyclic AMP for producing astrocyte-like cells.

[0010] The present invention can also be said to include the following aspects. [P1] A method for producing astrocytes, comprising: (A) upregulating transcription factors, including SRY-Box Transcription Factor 9 (SOX9), nuclear factor IA (NFIA), and nuclear factor IB (NFIB), in human pluripotent stem cells; and (B) culturing the human pluripotent stem cells in which the transcription factors are upregulated, thereby differentiating the human pluripotent stem cells into astrocytes. [P2] The method for producing astrocytes according to [P1], wherein in step (B), the human pluripotent stem cells are cultured in a pluripotent stem cell culture medium or a neuronal culture medium. [P3] The method for producing astrocytes described in [P1] or [P2], wherein step (A) is carried out by introducing a vector containing a nucleic acid consisting of a base sequence encoding the transcription factor into the human pluripotent stem cells. [P4] A method for producing astrocytes described in [P1] or [P2], wherein step (A) is carried out by adding or removing a tetracycline antibiotic to the culture medium of the human pluripotent stem cells into which a vector containing a nucleic acid consisting of a base sequence encoding the transcription factor under the control of a tetracycline response factor has been introduced. [P5] The method for producing astrocytes according to [P3] or [P4], wherein the vector contains a nucleic acid having a base sequence represented by the following formula (P1): F 1 -S 1 -F 2 -S 2 -F 3 …(P1) [In formula (1), F 1 , F 2 and F 3 represents, in no particular order, a nucleotide sequence encoding SOX9, a nucleotide sequence encoding NFIA, and a nucleotide sequence encoding NFIB; S 1 and S 2 represents the separator base sequence.] [P6] The method for producing astrocytes according to any one of [P3] to [P5], wherein the vector is a transposon vector. [P7] The method for producing astrocytes described in any of [P1] to [P6], wherein in step (B), the abundance of SOX9, NFIA, and NFIB in the human pluripotent stem cells is 1:1:1 in molar ratio. [P8] The method for producing astrocytes according to any one of [P1] to [P7], wherein the human pluripotent stem cells are human induced pluripotent stem cells. [P9] Astrocytes produced by the production method described in any one of [P1] to [P8]. [P10] Co-culture of astrocytes and neurons as described in [P9]. [P11] Human pluripotent stem cells for producing astrocytes, into which nucleic acids consisting of base sequences encoding transcription factors including SOX9, NFIA, and NFIB have been introduced. [P12] Human pluripotent stem cells for producing astrocytes according to [P11], in which a nucleic acid consisting of a base sequence encoding the transcription factor is introduced under the control of a tetracycline response factor. [Effects of the Invention]

[0011] According to the present invention, astrocyte-like cells can be efficiently produced in vitro. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram illustrating an outline of Experimental Example 1. [Figure 2] 1(a) and 1(b) are micrographs showing the results of Experimental Example 2. [Figure 3] 10(a) to 10(e) are fluorescence micrographs showing the results of Experimental Example 3. [Figure 4] 2(a) and 2(b) are graphs showing the results of quantitative RT-PCR in Experimental Example 4. [Figure 5] 10(a) and 10(b) are graphs showing the results of quantitative RT-PCR in Experimental Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0013] [Method of producing astrocyte-like cells] In one embodiment, the present invention provides a method for producing astrocyte-like cells, comprising: (A) upregulating transcription factors, including SOX9, NFIA, and NFIB, in human pluripotent stem cells; and (B) culturing the human pluripotent stem cells in which the transcription factors are upregulated, thereby differentiating the human pluripotent stem cells into astrocyte-like cells.

[0014] As used herein, astrocyte-like cells refer to cells that are functionally and morphologically equivalent to in vivo astrocytes, and may also be referred to as astrocytes. Astrocyte-like cells express astrocyte markers, which will be described later.

[0015] As will be described later in the Examples, the production method of this embodiment allows for efficient production of astrocyte-like cells in a short period of time. For example, five days after the initiation of transcription factor upregulation, cells exhibiting astrocyte-like morphology under microscopic observation can be obtained.

[0016] Furthermore, a high efficiency of astrocyte differentiation means that the expression levels of astrocyte markers, such as glial fibrillary acidic protein (GFAP) and S100 calcium binding protein B (S100β), are higher than those of controls.

[0017] Controls include cells in which none of SOX9, NFIA, and NFIB are upregulated. Specific examples include cells in which only one of SOX9, NFIA, and NFIB is upregulated, cells in which only SOX9 and NFIA are upregulated, cells in which only SOX9 and NFIB are upregulated, and cells in which only NFIA and NFIB are upregulated. As described later in the Examples, astrocytes obtained by the method of this embodiment, which upregulates SOX9, NFIA, and NFIB, have approximately 4- to 7-fold higher expression levels of astrocyte markers than those obtained when only SOX9 and NFIB are upregulated.

[0018] In step (A), transcription factors including SOX9, NFIA, and NFIB are upregulated in human pluripotent stem cells. Here, "upregulation" means increasing the amount present in the cells. Upregulation of SOX9, NFIA, and NFIB may involve, for example, introducing SOX9, NFIA, and NFIB into cells in the form of proteins. Alternatively, it may involve introducing SOX9, NFIA, and NFIB into cells in the form of mRNA. Alternatively, it may involve introducing expression vectors for the SOX9 gene, NFIA gene, and NFIB gene into cells. Alternatively, SOX9, NFIA, and NFIB may be upregulated by preparing cells transfected with expression vectors capable of controlling the expression of the SOX9 gene, NFIA gene, and NFIB gene, and inducing their expression.

[0019] Proteins, mRNA, and expression vectors can be introduced by commonly used methods as needed, such as electroporation, lipofection, and microinjection.

[0020] The expression vector may be a plasmid vector, a transposon vector, a viral vector, or a combination of these.

[0021] The transposon-based vector may be one that can be completely removed from cells as needed, such as the PiggyBac vector.

[0022] Examples of viral vectors include retroviral vectors, lentiviral vectors, adeno-associated viral vectors, adenoviral vectors, etc. When the expression vector is a viral vector, it can be introduced into cells by infecting them.

[0023] An example of an expression vector capable of controlling expression is one that can control expression in response to an external stimulus. Such an expression vector contains a promoter that can induce expression of downstream genes in response to an external stimulus, and at least the SOX9 gene, the NFIA gene, and the NFIB gene, the expression of which is controlled by the promoter.

[0024] The promoter is not particularly limited as long as it can control the expression of a downstream gene in response to an external stimulus. For example, when the external stimulus is the presence or absence of a tetracycline antibiotic, a promoter having a tetracycline response element (TRE) can be used.

[0025] In this case, step (A) is carried out by adding or removing a tetracycline antibiotic to the culture medium of the human pluripotent stem cells into which a vector containing the SOX9 gene, NFIA gene, and NFIB gene under the control of a tetracycline response element has been introduced.

[0026] When the external stimulus is the presence of a tetracycline antibiotic (Tet-On system), a complex of the tetracycline antibiotic and a reverse tetracycline-controlled transactivator (rtTA) binds to the TRE, thereby inducing the expression of downstream genes.

[0027] On the other hand, in the absence of tetracycline antibiotics (Tet-Off system), the tetracycline-controlled transactivator (tTA) binds to the TRE, thereby inducing the expression of downstream genes. In this case, in the presence of tetracycline antibiotics, the tetracycline antibiotic and tTA form a complex, which prevents the tTA from binding to the TRE, thereby suppressing the expression of downstream genes.

[0028] Examples of tetracycline antibiotics include tetracycline and tetracycline derivatives such as doxycycline. When doxycycline is used as the tetracycline antibiotic, the concentration of doxycycline added to the medium can be 0.1 to 10 μg / mL, and more preferably 1 to 2 μg / mL.

[0029] In addition, when the external stimulus is the presence of ecdysteroids, examples of promoters include those that can induce the expression of downstream genes upon binding of ecdysteroids to the ecdysone receptor-retinoid receptor complex. Examples of ecdysteroids include ecdysone, muristerone A, and ponasterone A.

[0030] Another example is a promoter that can induce downstream gene expression when the external stimulus is the presence of FKCsA by binding of FKCsA to a complex of the Gal4 DNA-binding domain fused to FKBP12 and the VP16 activator domain fused to cyclophilin.

[0031] The expression vector may, as necessary, contain an enhancer, a silencer, a drug selection marker, a replication origin, etc. Examples of drug selection markers include a hygromycin resistance gene, a puromycin resistance gene, and a neomycin resistance gene.

[0032] The NCBI accession numbers for the SOX9 protein are NP_000337.1, etc. The NCBI accession numbers for the SOX9 cDNA are NM_000346.4, etc. The NCBI accession numbers for the NFIA protein are NP_005586.1, NP_001128145.1, NP_001138984.1, etc. The NCBI accession numbers for the NFIA cDNA are NM_001134673.4, NM_001145511.2, NM_001145512.2, etc. The NCBI accession numbers for the NFIB protein are NP_001356409.1, NP_001356387.1, NP_001356397.1, etc. The NCBI accession numbers for NFIB cDNA are NM_001190737.2, NM_001190738.1, NM_001282787.1, etc.

[0033] SOX9, NFIA, and NFIB may have a mutation as long as they have the activity of inducing differentiation of human pluripotent stem cells into astrocyte-like cells. When SOX9, NFIA, or NFIB has a mutation, it preferably has 80% or more sequence identity, more preferably 90% or more sequence identity, and even more preferably 95% or more sequence identity to the protein or cDNA identified by the above-mentioned NCBI accession number.

[0034] Here, the sequence identity of an amino acid sequence is a value indicating the percentage of identity between a target amino acid sequence (target amino acid sequence) and a reference amino acid sequence (reference amino acid sequence). The sequence identity of a target amino acid sequence to a reference amino acid sequence can be determined, for example, as follows: First, the reference amino acid sequence and the target amino acid sequence are aligned. Here, gaps may be included in each amino acid sequence to maximize sequence identity. Next, the number of identical amino acids in the reference amino acid sequence and the target amino acid sequence is calculated, and the sequence identity can be determined according to the following formula (F1). Sequence identity (%) = number of matching amino acids / total number of amino acids in the target amino acid sequence × 100 ... (F1)

[0035] Similarly, the sequence identity of a subject base sequence to a reference base sequence can be determined, for example, as follows: First, the reference base sequence and the subject base sequence are aligned. Here, gaps may be included in each base sequence to maximize sequence identity. Next, the number of matching bases in the reference base sequence and the subject base sequence is calculated, and the sequence identity can be determined according to the following formula (F2). Sequence identity (%) = number of matched bases / total number of bases in the target sequence × 100 ... (F2)

[0036] In the production method of this embodiment, examples of pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). Furthermore, the pluripotent stem cells may be derived from healthy individuals or from patients with neurological disorders. When astrocyte-like cells are produced from pluripotent stem cells derived from patients with neurological disorders, the resulting astrocyte-like cells can be used as a model for neurological disorders. Such astrocyte-like cells are useful for elucidating the mechanisms of neurological disorders.

[0037] A vector that forcibly expresses the SOX9 gene, the NFIA gene, and the NFIB gene, or a vector that can induce the expression of the SOX9 gene, the NFIA gene, and the NFIB gene, may contain a nucleic acid consisting of a base sequence represented by the following formula (1): F 1 -S 1 -F 2 -S 2 -F 3 …(1)

[0038] In formula (1), F 1 , F 2 and F 3 represents, in no particular order, a nucleotide sequence encoding SOX9, a nucleotide sequence encoding NFIA, and a nucleotide sequence encoding NFIB; S 1 and S 2 represents the separator base sequence.

[0039] F 1 , F2 , F 3 may be nucleotide sequences encoding SOX9, NFIA, and NFIB, or may be nucleotide sequences encoding SOX9, NFIB, and NFIA, or may be nucleotide sequences encoding NFIA, NFIB, and SOX9, or may be nucleotide sequences encoding NFIA, SOX9, and NFIB, or may be nucleotide sequences encoding NFIB, SOX9, and NFIA, or may be nucleotide sequences encoding NFIB, NFIA, and SOX9, respectively.

[0040] Also, S 1 and S 2 may be the same or different nucleotide sequences. In formula (1), the separator nucleotide sequence is a nucleotide sequence that enables bicistronic expression, and examples thereof include an internal ribosome entry site (IRES) sequence and a 2A sequence. Examples of the 2A sequence include a T2A sequence derived from Thosea asigne, a P2A sequence derived from Porcine teschovirus, an F2A sequence derived from foot-and-mouth disease virus, and an E2A sequence derived from equine rhinitis A virus. The 2A sequence is also called a self-cleaving peptide sequence.

[0041] Bicistronic expression refers to the expression of two or more proteins from a single mRNA, resulting in a molar ratio of 1:1:1 for SOX9, NFIA, and NFIB.

[0042] Furthermore, it is possible to adjust the expression levels of SOX9, NFIA, and NFIB to a molar ratio of 1:1:1 without performing bicistronic expression, for example, by introducing equimolar amounts of SOX9, NFIA, and NFIB into cells, or by introducing equal copies of expression vectors that express SOX9, NFIA, and NFIB individually into cells.

[0043] Next, in step (B), human pluripotent stem cells with upregulated SOX9, NFIA, and NFIB are cultured. As a result, the human pluripotent stem cells differentiate into astrocyte-like cells. Since astrocytes are a type of glial cell, it is considered that a glial cell culture medium is used as the culture medium in step (B).

[0044] However, as described later in the Examples, the inventors have demonstrated that performing step (B) using a pluripotent stem cell culture medium or a neuronal culture medium results in a higher expression level of astrocyte markers than when using a glial cell culture medium. In other words, performing step (B) using a pluripotent stem cell culture medium or a neuronal culture medium enables efficient production of astrocyte-like cells.

[0045] Examples of pluripotent stem cell culture media include media commonly used for culturing iPS cells, ES cells, etc. More specifically, examples include Stem Fit (AK02N) (Ajinomoto Co., Inc.), Stem Fit (AK03) (Ajinomoto Co., Inc.), Cellartis DEF-CS 500 (Takara Bio Inc.), mTeRR1 (Stem Cell Technologies, Inc.), mTeSR-E8 (Stem Cell Technologies, Inc.), and Essential 8 (Thermo Fisher Scientific). Additives may be added to the pluripotent stem cell culture media. Examples of additives include basic fibroblast growth factor 2 (FGF2).

[0046] Examples of media for neuronal culture include media commonly used for neuronal culture. More specifically, examples include KBM Neural Stem (Kohjin Bio), Neurobasal Medium (Thermo Fisher Scientific), Neurobasal Plus Medium (Thermo Fisher Scientific), and Brain Phys (Stem Cell Technologies). Neuronal culture media may contain additives. Examples of additives include B27 supplement (Thermo Fisher Scientific), B27 Plus supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), SM1 supplement (Veritas), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin 3 (NT3), and dibutyryl cyclic AMP (dbcAMP).

[0047] The manufacturing method of this embodiment may include a step of differentiating human pluripotent stem cells into neural stem cells, or may not include a step of differentiating human pluripotent stem cells into neural stem cells, and may directly induce the differentiation of human pluripotent stem cells into astrocyte-like cells.

[0048] When the manufacturing method of this embodiment includes a step of differentiating human pluripotent stem cells into neural stem cells, the method may first involve inducing the differentiation of human pluripotent stem cells into neural stem cells, followed by a step (A) of upregulating transcription factors including SOX9, NFIA, and NFIB in the cells induced to differentiate into neural stem cells, and a step (B) of culturing the cells in which the transcription factors are upregulated, thereby differentiating the cells into astrocyte-like cells.

[0049] The step of differentiating human pluripotent stem cells into neural stem cells can be carried out by any conventional method, such as culturing human pluripotent stem cells in the presence of fibroblast growth factor 2 (FGF2), a Rho-associated protein kinase (ROCK) signaling pathway inhibitor, or leukemia inhibitory factor (LIF).

[0050] Examples of ROCK signaling pathway inhibitors include Y-27632 (CAS number: 129830-38-2), Fasudil / HA1077 (CAS number: 105628-07-7), H-1152 (CAS number: 871543-07-6), Wf-536 (CAS number: 539857-64-2), and derivatives thereof.

[0051] The final concentration of the ROCK signaling pathway inhibitor in the medium is usually 0.1 μM to 100 μM. The concentration is preferably 5 μM to 50 μM, and more preferably 10 μM to 30 μM.

[0052] [Astrocyte-like cells] In one embodiment, the present invention provides astrocyte-like cells produced by the above-described production method. The astrocyte-like cells of this embodiment can be efficiently produced in vitro, and therefore are suitable for use in basic research, elucidation of nervous system diseases, and the like.

[0053] The astrocyte-like cells of this embodiment may have exogenous SOX9, NFIA, and NFIB genes introduced into their genomes, and the SOX9, NFIA, and NFIB genes may be linked via a separator nucleotide sequence.

[0054] [Co-culture] In one embodiment, the present invention provides a co-culture of astrocyte-like cells and neuron-like cells as described above.

[0055] As used herein, neuron-like cells are cells that are functionally and morphologically equivalent to in vivo neurons, and can also be referred to as neurons. Neuron-like cells express neuron markers such as microtubule-associated protein 2 (MAP2), synapsin 1 (SYN1), and βIII-tubulin (TUBB3).

[0056] As described above, brain organoids prepared from pluripotent stem cells are primarily composed of neurons and neural stem cells, and do not contain fully mature glial cells. In contrast, the co-culture of this embodiment allows for the co-culture of large quantities of astrocyte-like cells and neuron-like cells, enabling the analysis of neuronal function under conditions similar to those in vivo.

[0057] [Use of medium for producing astrocyte-like cells] In one embodiment, the present invention provides use of a medium containing basic fibroblast growth factor or a medium containing at least one factor selected from the group consisting of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, neurotrophin 3, and dibutyryl cyclic AMP for producing astrocyte-like cells. As described above, the use of this embodiment enables efficient in vitro production of astrocyte-like cells. [Example]

[0058] The present invention will now be described in more detail with reference to experimental examples, but the present invention is not limited to these examples.

[0059] [Experimental Example 1] (Differentiation of iPS cells into astrocyte-like cells) Human iPS cells were transfected with SOX9, NFIA, and NFIB to investigate their differentiation into astrocyte-like cells. Figure 1 outlines the experimental setup. First, a reverse tetracycline-controlled transactivator (rtTA3G, VectorBuilder) was introduced into human iPS cells (1210B2 cell line) using a transposon vector (PiggyBac vector, VectorBuilder), and transgenic lines were obtained by drug selection. The hygromycin resistance gene was used as a drug selection marker for rtTA3G. An internal ribosome entry site (IRES) was inserted between the rtTA3G and hygromycin resistance genes to allow bicistronic expression of these genes. HyPBase (VectorBuilder) was used as the transposase.

[0060] Next, using a lentiviral vector (VectorBuilder), the SOX9, NFIB, and NFIA genes were introduced into a cell line stably expressing rtTA3G under the control of tetracycline response elements, and transgenic lines were obtained by drug selection. A puromycin resistance gene was used as a drug selection marker. A T2A sequence was inserted between the SOX9, NFIB, and NFIA genes to allow bicistronic expression of these genes. In addition, a nucleotide sequence encoding a FLAG tag was added to the SOX9 gene.

[0061] For comparison, we used a lentiviral vector (VectorBuilder) to introduce the SOX9 and NFIB genes into a cell line stably expressing rtTA3G under the control of tetracycline response elements, and then isolated transgenic lines by drug selection. A puromycin resistance gene was used as a drug selection marker. A T2A sequence was inserted between the SOX9 and NFIB genes to allow bicistronic expression of these genes. A nucleotide sequence encoding a FLAG tag was also added to the SOX9 gene.

[0062] The resulting transgenic lines were then expanded for 6 days in a pluripotent stem cell culture medium (product name: Stem Fit (AK02N), Ajinomoto Co., Inc.).

[0063] The cells were then dissociated and seeded onto new Matrigel-coated plates to induce differentiation into astrocyte-like cells. Doxycycline was added to the medium to induce expression of SOX9, NFIB, and NFIA genes, or SOX9 and NFIB genes. Culture media for pluripotent stem cells, glial cells, and neurons were used for comparison.

[0064] The medium used for pluripotent stem cell culture was a basal medium (product name "Stem Fit (AK02N) A solution", Ajinomoto Co., Inc.) supplemented with product names "Stem Fit (AK02N) B solution" (Ajinomoto Co., Inc.) and "Stem Fit (AK02N) C solution" (Ajinomoto Co., Inc.).

[0065] The glial cell culture medium used was a basal medium (trade name "KBM Neural Stem", Kohjin Bio Co., Ltd.) supplemented with 2% B27 supplement (Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 1% non-essential amino acids (NEAA) (Fujifilm Wako Pure Chemical Industries, Ltd.), 20 ng / mL fibroblast growth factor (FGF), 10 ng / mL epidermal growth factor (EGF), and 10 ng / mL neurotrophin 3 (NT3).

[0066] The medium used for neuronal culture was a basal medium (trade name "Neurobasal Plus Medium", Thermo Fisher Scientific) supplemented with 2% B27 supplement (Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 1% CultureOne supplement (Thermo Fisher Scientific), 200 μM ascorbic acid, 20 ng / mL brain-derived neurotrophic factor (BDNF), 20 ng / mL glial cell line-derived neurotrophic factor (GDNF), 20 ng / mL neurotrophin 3 (NT3), and 100 μM dibutyryl cyclic AMP (dbcAMP).

[0067] [Experimental Example 2] (Microscopic observation of astrocyte-like cells) Cells induced to differentiate into astrocyte-like cells in Experimental Example 1 were observed under a microscope. Figures 2(a) and (b) are photographs showing the results of microscopic observation of cells cultured for 5 days in the presence or absence of doxycycline in Experimental Example 1. These are representative results obtained when a neuronal culture medium was used. The scale bar is 100 μm.

[0068] Figure 2(a) is a photograph of cells cultured in the absence of doxycycline, and Figure 2(b) is a photograph of cells cultured in the presence of doxycycline. The photographs on the right in Figures 2(a) and (b) are enlarged views of the boxed areas in the photographs on the left.

[0069] As a result, it was revealed that cells in which expression of the SOX9 gene, NFIB gene, and NFIA gene was induced in the presence of doxycycline exhibited morphology characteristic of astrocytes, even just 5 days after the start of expression induction.

[0070] [Experimental Example 3] (Immunochemical staining study) The cells induced to differentiate into astrocyte-like cells in Experimental Example 1 were fixed and subjected to immunochemical staining. Figures 3(a) to (e) are fluorescence micrographs showing the results of immunochemical staining of cells cultured for 20 days in the presence of doxycycline, which were then fixed with paraformaldehyde. These are representative results obtained when a neuronal culture medium was used. The scale bar is 100 μm.

[0071] Figure 3(a) shows the results of nuclei stained with Hoechst 33342. Figure 3(b) shows the results of staining the astrocyte marker GFAP with an anti-GFAP antibody. Figure 3(c) shows the results of staining the FLAG tag with an anti-FLAG antibody. As mentioned above, the SOX9 gene contained a base sequence encoding the FLAG tag, allowing SOX9 expression to be detected using an anti-FLAG antibody. Figure 3(d) is a composite photograph of Figures 3(b) and (c). Figure 3(e) is an enlarged photograph of the boxed area in Figure 3(d). As a result, GFAP expression was confirmed, demonstrating astrocyte differentiation.

[0072] [Experimental Example 4] (Examination by quantitative RT-PCR) In Experimental Example 1, the mRNA of astrocyte markers in each cell line cultured for 20 days in the presence of doxycycline was quantified by quantitative RT-PCR. GFAP and S100β were examined as astrocyte markers. Cells were grown in a neuronal culture medium.

[0073] Figures 4(a) and (b) are graphs showing the results of quantitative RT-PCR. Figure 4(a) shows the results for GFAP, and Figure 4(b) shows the results for S100β. In Figures 4(a) and (b), "(-)" indicates the results for cells in which the expression of the SOX9 gene and NFIB gene was induced, and "NFIA" indicates the results for cells in which the expression of the SOX9 gene, NFIA gene, and NFIB gene was induced. The vertical axis indicates the mRNA expression level, expressed as a relative value, with the results for cells in which the expression of the SOX9 gene and NFIB gene was induced set at 1.

[0074] As a result, it was revealed that cells in which the expression of the SOX9 gene, NFIA gene, and NFIB gene was induced had more than four times the expression level of GFAP and approximately seven times the expression level of S100β compared to cells in which the expression of the SOX9 gene and NFIB gene was induced.

[0075] [Experimental Example 5] (Consideration of culture medium) In Experimental Example 1, cells were cultured for 20 days in the presence of doxycycline, and the expression of the SOX9, NFIA, and NFIB genes was induced. The mRNA levels of astrocyte markers in each cell were quantified by quantitative RT-PCR. GFAP and S100β were examined as astrocyte markers. Culture media for pluripotent stem cells, glial cells, and neurons were used.

[0076] Figures 5(a) and (b) are graphs showing the results of quantitative RT-PCR. Figure 5(a) shows the results for GFAP, and Figure 5(b) shows the results for S100β. In Figures 5(a) and (b), "PSC medium" shows the results when using a pluripotent stem cell culture medium, "Glial medium" shows the results when using a glial cell culture medium, and "Neuronal medium" shows the results when using a neuronal cell culture medium. The vertical axis shows the mRNA expression level, expressed as a relative value where the results when using a pluripotent stem cell culture medium are set to 1.

[0077] As a result, it was revealed that the expression level of astrocyte markers was higher when cultured in pluripotent stem cell culture medium or neuronal culture medium than in glial cell culture medium, which is thought to be more suitable for culturing astrocytes. [Industrial Applicability]

[0078] According to the present invention, astrocyte-like cells can be efficiently produced in vitro.

Claims

1. (A) upregulating transcription factors including SRY-Box Transcription Factor 9 (SOX9), nuclear factor IA (NFIA), and nuclear factor IB (NFIB) in human pluripotent stem cells; A method for producing astrocyte-like cells, comprising the step (B) of culturing the human pluripotent stem cells in which the transcription factor is upregulated, thereby differentiating the human pluripotent stem cells into astrocyte-like cells.

2. The method for producing astrocyte-like cells according to claim 1 , wherein in step (B), the human pluripotent stem cells are cultured in a pluripotent stem cell culture medium or a neuronal culture medium.

3. 3. The method for producing astrocyte-like cells according to claim 1 or 2, wherein step (A) is carried out by introducing a vector containing a nucleic acid encoding the transcription factor into the human pluripotent stem cells.

4. 3. The method for producing astrocyte-like cells according to claim 1 or 2, wherein step (A) is carried out by adding or removing a tetracycline antibiotic to a culture medium for the human pluripotent stem cells into which a vector containing a nucleic acid encoding the transcription factor under the control of a tetracycline response factor has been introduced.

5. The method for producing astrocyte-like cells according to claim 3 or 4, wherein the vector comprises a nucleic acid consisting of a base sequence represented by the following formula (1): F １ -S １ -F ２ -S ２ -F ３ …(1) [In formula (1), F １ , F ２ and F ３ represents, in no particular order, a nucleotide sequence encoding SOX9, a nucleotide sequence encoding NFIA, and a nucleotide sequence encoding NFIB; S １ and S ２ represents the separator base sequence.]

6. The method for producing astrocyte-like cells according to any one of claims 3 to 5, wherein the vector is a transposon vector.

7. The method for producing astrocyte-like cells according to any one of claims 1 to 6, wherein in step (B), the amounts of SOX9, NFIA, and NFIB present in the human pluripotent stem cells are in a molar ratio of 1:1:

1.

8. The method for producing astrocyte-like cells according to any one of claims 1 to 7, wherein the human pluripotent stem cells are human induced pluripotent stem cells.

9. The medium for culturing pluripotent stem cells comprises basic fibroblast growth factor, 3. The method for producing astrocyte-like cells according to claim 2, wherein the neuronal culture medium contains at least one factor selected from the group consisting of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, neurotrophin 3, and dibutyryl cyclic AMP.

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