Method for producing oligodendrocyte-like cells

Abstract

This method for producing oligodendrocyte-like cells includes: a step (A) for increasing the presence of an oligodendrocyte transcription factor 2 (OLIG2) variant and SRY-box transcription factor 10 (SOX10) in human pluripotent stem cells; and a step (B) for culturing the human pluripotent stem cells in which the presence of the OLIG2 variant and SOX 10 has increased, and, consequently, differentiating the human pluripotent stem cells into oligodendrocyte-like cells. The OLIG2 variant is deficient in the serine residue at position 147 of wild-type OLIG2, or the serine residue at position 147 of wild-type OLIG2 has been replaced by an amino acid other than serine.

Description

Technical Field

[0001] The present invention relates to a method for producing oligodendrocyte-like cells. More specifically, the present invention relates to a method for producing oligodendrocyte-like cells, oligodendrocyte-like cells, and a co-culture of oligodendrocyte-like cells and neuron-like cells. This application claims priority based on Japanese Patent Application No. 2020-152305 filed in Japan on September 10, 2020, and the contents thereof are incorporated herein by reference.

Background Art

[0002] There is a need to use nervous system tissues for basic research and elucidation of nervous system diseases. However, experimental results obtained using nervous system tissues of experimental animals such as mice and rats may have problems with extrapolation to humans, and there are limitations in using human nervous system tissues. Therefore, forming and using human nervous system tissues in vitro has been considered.

[0003] However, it is known that brain organoids prepared from pluripotent stem cells such as induced pluripotent stem cells (iPS) cells are mainly composed of neurons and neural stem cells and do not contain sufficiently mature glial cells (astrocytes, oligodendrocytes, microglia, etc.).

[0004] Glial cells are responsible for the survival and functional expression of neurons, such as supplying nutrients to neurons. It has become clear that not only neurons but also the role of glial cells is important for the functional expression of the brain. Therefore, a technique for producing glial cells in vitro is required.

[0005] For example, Non-Patent Document 1 describes that human iPS cells were induced to differentiate into oligodendrocyte-like cells by expressing the SRY-Box Transcription Factor 10 (SOX10) gene.

[0006] Furthermore, Non-Patent Document 2 states that OLIG2 expression is necessary for the differentiation of human ES cells into oligodendrocyte-like cells. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Garcia-Leon JA, et al., SOX10 Single Transcription Factor-Based Fast and Efficient Generation of Oligodendrocytes from Human Pluripotent Stem Cells, Stem Cell Reports, 10(2), 655-672, 2018. [Non-Patent Document 2] Hu, BY, et al., Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects, Development, 136(9), 1443-1452, 2009. [Overview of the project] [Problems that the invention aims to solve]

[0008] However, the inventors found that when iPS cells were differentiated into oligodendrocyte-like cells using the method described in Reference 1, there was a large difference in differentiation efficiency among iPS cell lines, and that differentiation occurred not only into oligodendrocyte-like cells but also into astrocyte-like cells. Furthermore, the method described in Reference 2 resulted in differentiation of motor neurons in addition to oligodendrocyte-like cells. Therefore, the present invention aims to provide a technology for efficiently producing oligodendrocyte-like cells in vitro. [Means for solving the problem]

[0009] The present invention includes the following embodiments. [1] A method for producing oligodendrocyte-like cells, comprising the steps of (A) increasing the abundance of an oligodendrocyte Transcription Factor 2 (OLIG2) mutant and SRY-Box Transcription Factor 10 (SOX10) in human pluripotent stem cells, and (B) culturing the human pluripotent stem cells in which the abundance of the OLIG2 mutant and SOX10 has increased, thereby differentiating the human pluripotent stem cells into oligodendrocyte-like cells, wherein the OLIG2 mutant is either deficient in the 147th serine residue of wild-type OLIG2, or the 147th serine residue of wild-type OLIG2 is substituted with an amino acid other than serine. [2] The method for producing oligodendrocyte-like cells according to [1], wherein step (A) is performed by introducing a vector containing nucleic acids encoding the OLIG2 mutant and SOX10 into the human pluripotent stem cells. [3] The method for producing oligodendrocyte-like cells according to [1], wherein step (A) is performed by adding or removing a tetracycline antibiotic to the culture medium of human pluripotent stem cells into which a vector containing the OLIG2 mutant and the nucleic acid encoding SOX10 has been introduced, under the control of a tetracycline responsive factor. [4] A method for producing oligodendrocyte-like cells according to [2] or [3], wherein the vector containing the nucleic acid encoding the OLIG2 variant and SOX10 is a combination of the vector containing the nucleic acid encoding the OLIG2 variant and the vector containing the nucleic acid encoding SOX10. [5] The method for producing oligodendrocyte-like cells according to [4], wherein the vector containing the nucleic acid encoding the OLIG2 mutant is a transposon vector. [6] A method for producing oligodendrocyte-like cells according to [4] or [5], wherein the vector containing the nucleic acid encoding SOX10 is a lentiviral vector. [7] A method for producing oligodendrocyte-like cells according to any one of [1] to [6], wherein the human pluripotent stem cells are human induced pluripotent stem cells. Oligodendrocyte-like cells produced by any of the manufacturing methods described in [8][1] to [7]. Co-cultures of oligodendrocyte-like cells and neuronal-like cells as described in [9][8].

[0010] The present invention can also be said to include the following embodiments. [P1] A method for producing oligodendrocytes, comprising the steps of (A) increasing the abundance of an oligodendrocyte Transcription Factor 2 (OLIG2) mutant and SRY-Box Transcription Factor 10 (SOX10) in human pluripotent stem cells, and (B) culturing the human pluripotent stem cells in which the abundance of the OLIG2 mutant and SOX10 has increased, thereby differentiating the human pluripotent stem cells into oligodendrocytes, wherein the OLIG2 mutant is either deficient in the 147th serine residue of wild-type OLIG2, or in which the 147th serine residue of wild-type OLIG2 is substituted with an amino acid other than serine. [P2] The method for producing oligodendrocytes according to [P1], wherein step (A) is performed by introducing a vector containing nucleic acids comprising the OLIG2 mutant and the base sequence encoding SOX10 into the human pluripotent stem cells. [P3] The method for producing oligodendrocytes according to [P1], wherein step (A) is performed by adding or removing a tetracycline antibiotic to the culture medium of human pluripotent stem cells into which a vector comprising the OLIG2 mutant and the base sequence encoding SOX10 has been introduced, under the control of a tetracycline responsive factor. [P4] A method for producing oligodendrocytes according to [P2] or [P3], wherein the vector containing nucleic acids comprising the nucleotide sequence encoding the OLIG2 variant and SOX10 is a combination of the vector containing nucleic acids comprising the nucleotide sequence encoding the OLIG2 variant and the vector containing nucleic acids comprising the nucleotide sequence encoding SOX10. [P5] A method for producing oligodendrocytes according to [P4], wherein the vector containing nucleic acid comprising the base sequence encoding the OLIG2 mutant is a transposon vector. [P6] A method for producing oligodendrocytes according to [P4] or [P5], wherein the vector containing nucleic acid comprising the base sequence encoding SOX10 is a lentiviral vector. [P7] A method for producing oligodendrocytes according to any one of [P1] to [P6], wherein the human pluripotent stem cells are human induced pluripotent stem cells. Oligodendrocytes manufactured by the manufacturing method described in any of the following sections: [P8], [P1], or [P7]. Co-culture of oligodendrocytes and nerve cells as described in [P9][P8]. Human pluripotent stem cells for oligodendrocyte production, into which nucleic acids comprising the [P10]OLIG2 mutant and the nucleotide sequence encoding SOX10 are introduced, wherein the OLIG2 mutant is either deficient in the 147th serine residue of wild-type OLIG2, or the 147th serine residue of wild-type OLIG2 is substituted with an amino acid other than serine. [P11] Human pluripotent stem cells for oligodendrocyte production according to [P10], wherein nucleic acids comprising the OLIG2 mutant and the nucleotide sequence encoding SOX10 are introduced under the control of a tetracycline response factor. [Effects of the Invention]

[0011] According to the present invention, a technology for efficiently producing oligodendrocyte-like cells in vitro can be provided. [Brief explanation of the drawing]

[0012] [Figure 1] (a) and (b) are graphs showing the results of quantitative RT-PCR in Experimental Example 1. [Figure 2] This is a diagram illustrating the outline of Experiment Example 2. [Figure 3] This is a fluorescence microscope image showing the results of Experimental Example 3. [Figure 4] (a) and (b) are graphs showing the results of quantitative RT-PCR in Experimental Example 4.

Mode for Carrying Out the Invention

[0013] [Method for Producing Oligodendrocyte-like Cells] In one embodiment, the present invention provides a method for producing oligodendrocyte-like cells, comprising: step (A) of increasing the abundance of an OLIG2 variant and SOX10 in human pluripotent stem cells; and step (B) of culturing the human pluripotent stem cells in which the abundance of the OLIG2 variant and SOX10 has been increased, resulting in the differentiation of the human pluripotent stem cells into oligodendrocyte-like cells, wherein the OLIG2 variant lacks the 147th serine residue of wild-type OLIG2 or has the 147th serine residue of wild-type OLIG2 substituted with an amino acid other than serine.

[0014] As used herein, oligodendrocyte-like cells mean cells that are functionally and morphologically equivalent to oligodendrocytes in vivo and can also be referred to as oligodendrocytes. Oligodendrocyte-like cells express oligodendrocyte markers described below.

[0015] As will be described later in the Examples, according to the production method of the present embodiment, oligodendrocyte-like cells can be efficiently produced in a short period of time. For example, 10 days after increasing the abundance of the OLIG2 variant and SOX10 in human pluripotent stem cells, cells showing a morphology characteristic of oligodendrocytes can be obtained by microscopic observation.

[0016] A high differentiation efficiency into oligodendrocyte-like cells means that the expression level of oligodendrocyte markers is high compared to the control, the expression levels of neural stem cell markers, neuronal markers, and astrocyte markers are low compared to the control, and the variation in the differentiation induction efficiency among human pluripotent stem cell lines is small.

[0017] Examples of oligodendrocyte markers include Platelet Derived Growth Factor Receptor Alpha (PDGFRA), Proteolipid Protein 1 (PLP1), and 2',3'-Cyclic Nucleotide 3' Phosphodiesterase (CNP).

[0018] Examples of neural stem cell markers include SRY (sex determining region Y)-box 2 (SOX2) and Paired box 6 (PAX6).

[0019] Examples of neuronal cell markers include microtubule-associated protein 2 (MAP2), synapsin 1 (SYN1), and βIII-tubulin (TUBB3).

[0020] Examples of astrocyte markers include Glial Fibrillary Acidic Protein (GFAP) and S100 Calcium Binding Protein B (S100β).

[0021] Examples of control groups include cells with increased levels of SOX10 only, cells with increased levels of wild-type OLIG2 only, and cells with increased levels of both wild-type OLIG2 and SOX10.

[0022] As described later in the examples, oligodendrocyte-like cells obtained by the method of this embodiment, which increases the abundance of OLIG2 mutants and SOX10, show approximately 3 to 5 times higher expression levels of oligodendrocyte markers compared to cases where only the abundance of SOX10 is increased. Furthermore, the expression levels of astrocyte markers are reduced to approximately 1 / 2 to 1 / 5. In addition, the variability in differentiation induction efficiency among human pluripotent stem cell lines is also reduced.

[0023] In step (A), the abundance of the OLIG2 mutant and SOX10 in human pluripotent stem cells is increased. Here, increasing the abundance may be done, for example, by introducing the OLIG2 mutant and SOX10 into cells in the form of proteins. Alternatively, the OLIG2 mutant and SOX10 may be introduced into cells in the form of mRNA. Alternatively, an expression vector that forces the expression of the gene encoding the OLIG2 mutant (a nucleic acid consisting of a nucleotide sequence encoding the OLIG2 mutant) and the gene encoding SOX10 (a nucleic acid consisting of a nucleotide sequence encoding SOX10) may be introduced into cells. Alternatively, the abundance of the OLIG2 mutant and SOX10 may be increased by preparing cells into which an expression vector capable of controlling the expression of the gene encoding the OLIG2 mutant and the gene encoding SOX10 has been introduced, and inducing their expression.

[0024] Here, the vector containing the nucleic acids encoding the OLIG2 variant and SOX10 may contain the nucleic acid encoding the OLIG2 variant and the nucleic acid encoding SOX10 within a single vector, or it may consist of a combination of multiple vectors, each containing the nucleic acid encoding the OLIG2 variant and the nucleic acid encoding SOX10.

[0025] The introduction of proteins, mRNA, and expression vectors can be carried out by commonly used methods as needed, such as electroporation, lipofection, and microinjection.

[0026] The expression vector may be a plasmid vector, a transposon vector, a viral vector, or a combination of these.

[0027] The transposon vector may be one that can be completely removed from the cell if necessary. Examples of such transposon vectors include the PiggyBac vector.

[0028] Examples of viral vectors include retroviral vectors, lentiviral vectors, adeno-associated virus vectors, and adenovirus vectors. When the expression vector is a viral vector, it can be introduced by infecting cells.

[0029] For example, as will be described later in the examples, a vector containing nucleic acid encoding the OLIG2 variant may be a transposon vector. Alternatively, a vector containing nucleic acid encoding SOX10 may be a lentiviral vector.

[0030] Examples of expression vectors whose expression can be controlled include those whose expression can be controlled in response to external stimuli. Such expression vectors include at least one promoter that can induce the expression of downstream genes in response to external stimuli, and the OLIG2 mutant gene and the SOX10 gene whose expression is controlled by the promoter.

[0031] The promoter is not particularly limited as long as it can regulate the expression of downstream genes in response to external stimuli. For example, when the external stimulus is the presence or absence of tetracycline antibiotics, a promoter having a tetracycline response factor (TRE) is an example.

[0032] In this case, step (A) is carried out by adding or removing a tetracycline antibiotic to the culture medium of human pluripotent stem cells into which a vector containing the OLIG2 mutant and the nucleic acid encoding SOX10 has been introduced, under the control of a tetracycline responsive factor.

[0033] When the external stimulus is the presence of a tetracycline antibiotic (Tet-On system), a complex of the tetracycline antibiotic and reverse tetracycline regulatory transactivator (rtTA) can bind to TRE, thereby inducing the expression of downstream genes.

[0034] On the other hand, when the external stimulus is in the absence of tetracycline antibiotics (Tet-Off system), tetracycline-regulating transactivators (tTAs) can induce the expression of downstream genes by binding to TREs. In this case, in the presence of tetracycline antibiotics, the tetracycline antibiotics and tTAs form a complex, preventing them from binding to TREs and thus suppressing the expression of downstream genes.

[0035] Examples of tetracycline antibiotics include tetracycline and tetracycline derivatives such as doxycycline. When using doxycycline as a tetracycline antibiotic, the concentration of doxycycline added to the culture medium can be 0.1 to 10 μg / mL, with 1 to 2 μg / mL being more preferable.

[0036] Furthermore, when the external stimulus is the presence of an ecdysteroid, a promoter that can induce the expression of downstream genes by binding the ecdysteroid to the ecdysone receptor-retinoid receptor complex is also an option. Examples of ecdysteroids include ecdysone, muristerone A, and ponasterone A.

[0037] Furthermore, in the case of an external stimulus being the presence of FKCsA, a promoter can be mentioned in which the expression of downstream genes can be induced by the binding of FKCsA to a complex of a Gal4 DNA-binding domain fused to FKBP12 and a VP16 activator domain fused to cyclophyllin.

[0038] The expression vector may optionally include enhancers, silencers, drug selection markers, origins of replication, etc. Examples of drug selection markers include hygromycin resistance genes, puromycin resistance genes, neomycin resistance genes, etc.

[0039] The NCBI accession number for wild-type human OLIG2 protein is NP_005797.1, etc. The NCBI accession number for wild-type human OLIG2 cDNA is NM_005806.4, etc.

[0040] It is known that when the 147th serine residue of wild-type human OLIG2 is phosphorylated, OLIG2 forms a homodimer. The OLIG2 mutant used in the production method of this embodiment can be any human OLIG2 that does not form a homodimer, and may be a mutant lacking the 147th serine residue of wild-type OLIG2, or a mutant in which the 147th serine residue of wild-type OLIG2 is replaced with an amino acid residue other than a serine residue. Alanine residues are preferred as the amino acid residue other than a serine residue.

[0041] As described later in the examples, the inventors discovered that increasing the expression level of the OLIG2 mutant together with SOX10 in human pluripotent stem cells allows for the efficient production of oligodendrocyte-like cells in a short period of time, thus completing the present invention.

[0042] The NCBI accession number for human SOX10 protein is NP_008872.1, etc. The NCBI accession number for human SOX10 cDNA is NM_006941.4, etc.

[0043] OLIG2 mutants may have mutations insofar as they have the activity to induce differentiation of human pluripotent stem cells into oligodendrocyte-like cells. If an OLIG2 mutant has further mutations in addition to a mutation that lacks the 147th serine residue of wild-type OLIG2, or a mutation in which the 147th serine residue of wild-type OLIG2 is replaced with an amino acid other than serine, it is preferable that it has 80% or more sequence identity with the wild-type protein or cDNA identified by the NCBI accession number described above, more preferably 90% or more sequence identity, and even more preferably 95% or more sequence identity.

[0044] Furthermore, SOX10 may have mutations insofar as it has the activity to induce differentiation of human pluripotent stem cells into oligodendrocyte-like cells. If SOX10 has mutations, it is preferable that it has 80% or more sequence identity with the wild-type protein or cDNA identified by the NCBI accession number described above, more preferably 90% or more sequence identity, and even more preferably 95% or more sequence identity.

[0045] Here, amino acid sequence identity is a value that indicates the proportion of the target amino acid sequence that matches a reference amino acid sequence. The sequence identity of the target amino acid sequence with respect to the 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 matching 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)

[0046] Similarly, the sequence identity of a target nucleotide sequence with respect to a reference nucleotide sequence can be determined, for example, as follows. First, the reference nucleotide sequence and the target nucleotide sequence are aligned. Here, gaps may be included in each nucleotide sequence to maximize sequence identity. Next, the number of matching nucleotides in the reference nucleotide sequence and the target nucleotide sequence is calculated, and the sequence identity can be determined according to the following formula (F2). Sequence identity (%) = Number of matching bases / Total number of bases in the target sequence × 100 …(F2)

[0047] In the manufacturing 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 diseases. When oligodendrocyte-like cells are produced from pluripotent stem cells derived from patients with neurological diseases, the obtained oligodendrocyte-like cells can be used as a model for neurological diseases. Such oligodendrocyte-like cells are useful for elucidating the mechanisms of neurological diseases.

[0048] Next, in step (B), human pluripotent stem cells with increased levels of OLIG2 mutant and SOX10 are cultured. As a result, the human pluripotent stem cells differentiate into oligodendrocyte-like cells.

[0049] The manufacturing method of this embodiment may include a step of differentiating human pluripotent stem cells into neural stem cells, or it may not include a step of differentiating human pluripotent stem cells into neural stem cells, and may directly induce differentiation of human pluripotent stem cells into oligodendrocyte-like cells.

[0050] If the manufacturing method of this embodiment includes a step of differentiating human pluripotent stem cells into neural stem cells, then the following steps may be taken: (A) first, inducing differentiation of human pluripotent stem cells into neural stem cells; and then increasing the amount of OLIG2 mutant and SOX10 in the cells differentiated into neural stem cells; and (B) culturing the cells in which the amount of OLIG2 mutant and SOX10 has increased, resulting in the cells differentiating into oligodendrocyte-like cells.

[0051] For the process of differentiating human pluripotent stem cells into neural stem cells, any commonly used method can be appropriately employed. For example, one method involves culturing human pluripotent stem cells in the presence of Fibroblast Growth Factor 2 (FGF2), a Rho-associated protein kinase (ROCK) signaling pathway inhibitor, and Leukemia Inhibitory Factor (LIF).

[0052] 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 their derivatives.

[0053] The final concentration of the ROCK signaling pathway inhibitor in the culture medium is typically 0.1 μM to 100 μM, preferably 5 μM to 50 μM, and more preferably 10 μM to 30 μM.

[0054] [Oligodendrocyte-like cells] In one embodiment, the present invention provides oligodendrocyte-like cells produced by the manufacturing method described above. Since the oligodendrocyte-like cells of this embodiment can be efficiently produced in vitro, they can be suitably used in basic research and in elucidating neurological diseases.

[0055] The oligodendrocyte-like cells of this embodiment may have a gene encoding an OLIG2 variant and a gene encoding an exogenous SOX10 introduced into their genome.

[0056] [Co-culture] In one embodiment, the present invention provides a co-culture of oligodendrocyte-like cells and nerve-like cells as described above.

[0057] In this specification, "neuron-like cells" refer to cells that are functionally and morphologically equivalent to nerve cells in the body, and can also be referred to as "nerve cells." Neuron-like cells express the nerve cell markers described above.

[0058] As mentioned above, brain organoids created from pluripotent stem cells are mainly composed of nerve cells and neural stem cells, and do not contain sufficiently mature glial cells. In contrast, the co-culture of this embodiment allows for the co-culture of a large quantity of oligodendrocyte-like cells and nerve-like cells, enabling the analysis of nerve cell function in a state close to that of a living organism. [Examples]

[0059] The present invention will now be described in more detail with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[0060] [Experimental Example 1] (Differentiation of iPS cells into oligodendrocyte-like cells 1) Human pluripotent stem cells were differentiated into oligodendrocyte-like cells using conventional methods. Specifically, human pluripotent stem cells were expressed with SOX10 and then differentiated into oligodendrocyte-like cells. The human pluripotent stem cells used were the iPS cell lines 1210B2, 414C2, and 201B7, and the ES cell line khES1.

[0061] First, a transposon vector (PiggyBac vector, Vectorbuilder Inc.) was used to introduce a reverse tetracycline-regulating transactivator (rtTA3G, Vectorbuilder Inc.) into human pluripotent stem cell lines (1210B2, 414C2, 201B7, khES1), and gene transfected cells were obtained through drug selection. The hygromycin resistance gene was used as the drug selection marker for rtTA3G. An internal ribosome entry site (IRES) sequence was introduced between rtTA3G and the hygromycin resistance gene, and these genes were expressed bicistronically. HyPBase (Vectorbuilder Inc.) was used as the transposase.

[0062] Next, each pluripotent stem cell line was dissociated into single cells and re-seeded. This point was designated as day 0. Subsequently, each pluripotent stem cell line was differentiated to form neural stem cells by suspension culture in the presence of Fibroblast Growth Factor 2 (FGF2), a Rho-associated protein kinase (ROCK) signaling pathway inhibitor, and Leukemia Inhibitory Factor (LIF), and these cells formed neurospheres.

[0063] Next, on day 14, the SOX10 gene was introduced into each neurosphere using a lentiviral vector (VectorBuilder) under the control of a tetracycline-responsive factor. Subsequently, the cells were dissociated and seeded onto new plates coated with Matrigel for culture. Doxycycline was added to the culture medium to induce SOX10 gene expression. The culture medium used was a glial cell-producing medium oriented for oligodendrocyte precursor differentiation. For comparison, cells cultured without the addition of doxycycline were also prepared.

[0064] Next, cells were harvested on days 40 and 60, and the expression levels of oligodendrocyte markers and astrocyte markers were quantified by quantitative RT-PCR. PDGFRA was used as the oligodendrocyte marker, and GFAP was used as the astrocyte marker.

[0065] Figures 1(a) and (b) are graphs showing the results of quantitative RT-PCR. Figure 1(a) shows the results of quantifying PDGFRA expression, and Figure 1(b) shows the results of quantifying GFAP expression. In Figures 1(a) and (b), "Day 40" indicates the result on day 40, "Day 60" indicates the result on day 60, "Dox-" indicates the result without doxycycline addition, and "Dox+" indicates the result with doxycycline addition.

[0066] As a result, as shown in Figure 1(a), it was confirmed that the addition of doxycycline to the culture medium induced the expression of oligodendrocyte markers. However, it became clear that there were significant differences in the expression levels of oligodendrocyte markers among the various pluripotent stem cell lines. Furthermore, as shown in Figure 1(b), an increase in the expression level of astrocyte markers was also confirmed. This result indicates that the induction of SOX10 gene expression induced differentiation not only in oligodendrocyte-like cells but also in astrocyte-like cells.

[0067] [Experimental Example 2] (Differentiation of iPS cells into oligodendrocyte-like cells 2) Human pluripotent stem cells were expressed with the OLIG2 mutant and SOX10, and then differentiated into oligodendrocyte-like cells. The human pluripotent stem cells used were the iPS cell lines 1210B2, 414C2, and 201B7.

[0068] Figure 2 illustrates the outline of the experiment. First, using a transposon vector (PiggyBac vector, Vectorbuilder Inc.), the human SOX10 gene was introduced into each iPS cell under the control of a reverse tetracycline-regulating transactivator (rtTA3G, Vectorbuilder Inc.) and a tetracycline-responsive factor, and gene-transduced cells were obtained by drug selection.

[0069] For rtTA3G, the hygromycin resistance gene was used as a drug selection marker. For the human SOX10 gene, the puromycin resistance gene was used as a drug selection marker. An internal ribosome entry site (IRES) sequence was introduced between rtTA3G and the hygromycin resistance gene, and these genes were expressed bicistronically. HyPBase (VectorBuilder, Inc.) was used as the transposase.

[0070] Next, using lentiviral vectors (VectorBuilder), the gene encoding the OLIG2 mutant was introduced into cell lines into which rtTA3G and SOX10 had been introduced, under the control of a tetracycline responsive factor, and gene-transformed cells were obtained by drug selection. The neomycin resistance gene was used as the drug selection marker.

[0071] As the gene encoding the OLIG2 mutant, we used a gene encoding a mutant of wild-type human OLIG2 in which the 147th serine residue was mutated to an alanine residue (hereinafter sometimes referred to as "Olig2S147A"). The OLIG2 mutant was then given a nucleotide sequence encoding an HA tag.

[0072] For comparison, iPS cells without the OLIG2 mutant were also prepared.

[0073] Next, the resulting gene-transformed cells were cultured for 6 days. The culture medium used was pluripotent stem cell culture medium (product name "Stem Fit (AK02N)", manufactured by Ajinomoto Co., Inc.).

[0074] Next, the cells were dissociated and seeded onto new plates coated with Matrigel to induce differentiation into oligodendrocyte-like cells. Doxycycline was also added to the culture medium to induce expression of the OLIG2 mutant and / or SOX10, or SOX10 alone. Neuronal cell culture medium was used as the culture medium. For neuronal cell culture, we used a culture medium consisting of a basic medium (product 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).

[0075] [Experimental Example 3] (Examination using immunochemical staining) Cells differentiated into oligodendrocyte-like cells in Experimental Example 2 were fixed and immunostained. Figure 3 is a representative fluorescence micrograph showing the results of immunostaining of cells (1210B2 strain) that were cultured for 10 days in the presence of doxycycline and then fixed with paraformaldehyde. This is a representative result using neuronal cell culture medium. The scale bar is 100 μm.

[0076] In Figure 3, "Hoechst" shows the result of staining the nucleus with Hoechst 33342, "HA" shows the result of staining the HA tag with an anti-HA antibody, "PDGFRα" shows the result of detecting the expression of the oligodendrocyte marker PDGFRA with an anti-PDGFRα antibody, and "Merge" is a composite image showing the detection of Hoechst 33342, HA, and PDGFRα.

[0077] Furthermore, "SOX10" indicates the result of SOX10 expression induction, "Olig2S147A" indicates the result of Olig2S147A expression induction, "-" indicates the result of not inducing Olig2S147A expression, and the arrowhead indicates the presence of a projection-like structure characteristic of oligodendrocytes.

[0078] As a result, it was revealed that when the OLIG2 mutant and SOX10 were expressed, the expression levels of oligodendrocyte markers increased compared to when SOX10 was expressed alone, and the morphological characteristics of oligodendrocytes were more pronounced.

[0079] [Experimental Example 4] (Study using quantitative RT-PCR) In Experimental Example 2, mRNA levels of oligodendrocyte and astrocyte markers were quantified by quantitative RT-PCR in each cell cultured for 10 days in the presence of doxycycline. PDGFRA was investigated as an oligodendrocyte marker, and GFAP was investigated as an astrocyte marker.

[0080] Figures 4(a) and (b) are graphs showing the results of quantitative RT-PCR. Figure 4(a) shows the results for PDGFRA, and Figure 4(b) shows the results for GFAP. In Figures 4(a) and (b), the vertical axis shows the mRNA expression level (relative value). "SOX10" indicates the result of SOX10 expression induction, "Olig2S147A" indicates the result of Olig2S147A expression induction, and "(-)" indicates the result of no Olig2S147A expression induction.

[0081] As a result, it was found that when OLIG2 mutants and SOX10 were expressed, the expression level of oligodendrocyte markers increased by approximately 3 to 5 times compared to when SOX10 was expressed alone, and that there was little variation in expression levels among cell lines.

[0082] Furthermore, it was revealed that when OLIG2 mutants and SOX10 were expressed, the expression levels of astrocyte markers were reduced to approximately 1 / 2 to 1 / 5 compared to when SOX10 was expressed alone, and the variability in expression levels among cell lines was also small. [Industrial applicability]

[0083] According to the present invention, a technology for efficiently producing oligodendrocyte-like cells in vitro can be provided.

Claims

1. Step (A) involves increasing the amount of Oligodendrocyte Transaction Factor 2 (OLIG2) mutant and SRY-Box Transaction Factor 10 (SOX10) in human induced pluripotent stem cells, The process includes (B) culturing the human induced pluripotent stem cells in which the amount of OLIG2 mutant and SOX10 has been increased, thereby differentiating the human induced pluripotent stem cells into oligodendrocyte-like cells. The OLIG2 mutant is characterized in which the 147th serine residue of wild-type OLIG2 is replaced with an alanine residue. A method for producing oligodendrocyte-like cells, wherein step (A) is performed by adding or removing a tetracycline antibiotic to a culture medium of human induced pluripotent stem cells into which a vector containing the OLIG2 mutant and the nucleic acid encoding SOX10 has been introduced, under the control of a tetracycline responsive factor.

2. A method for producing oligodendrocyte-like cells according to claim 1, wherein the vector containing the nucleic acid encoding the OLIG2 variant and SOX10 consists of a combination of the vector containing the nucleic acid encoding the OLIG2 variant and the vector containing the nucleic acid encoding SOX10.

3. A method for producing oligodendrocyte-like cells according to claim 1 or 2, wherein the vector containing the nucleic acid encoding the OLIG2 variant is a transposon vector.

4. A method for producing oligodendrocyte-like cells according to claim 1 or 2, wherein the vector containing the nucleic acid encoding SOX10 is a lentiviral vector.

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