JP6906231B2 - Astrocyte-like cells and their preparation method - Google Patents

Description

The present invention is a method for preparing astrocyte-like cells from human cells in vitro, an in vitro neural tissue model including astrocyte-like cells prepared by the method, astrocyte-like cells and neurons, derived from a patient with a nervous system disease. The present invention relates to a method for evaluating the efficacy or toxicity of a test substance against nervous system diseases using astrocyte-like cells, an inducer from human cells to astrocyte-like cells, and a kit for inducing human cells to astrocyte-like cells.

Astrocytes are nervous system cells that are gradually being recognized to play a major role in the pathophysiology of many nervous system diseases. (Non-Patent Document 1).

A rapid and large supply of human astrocytes is needed to study the pathophysiological role of astrocytes in nervous system diseases. However, at this time, the sources of human astrocytes are limited. It is ethically impossible to obtain astrocytes by biopsy from a living patient with a nervous system disease. Although astrocytes can be obtained from the postmortem brain of patients with nervous system diseases, mass supply is extremely difficult.

A method for preparing astrocytes by inducing differentiation from human induced pluripotent stem cells (iPS cells) has been reported (Non-Patent Document 2).

In addition, NFIA (Nuclear factor I / A), NFIB (Nuclear factor I / B) and SOX9 (SRY (sex determining region Y) -box 9) were introduced into mouse fibroblasts to introduce astrocyte-like cells. A method for directly inducing a mouse has been reported (Non-Patent Document 3). In the literature, attempts have been made to introduce the three factors into human fibroblasts and induce them into astrocyte-like cells.

It has been reported that knockdown of the p14ARF gene, p16INK4 gene, or p16INK4 gene present at the CDKN2A locus can improve the efficiency of reprogramming from human fibroblasts to iPS cells (Non-Patent Documents). 4). It has also been reported that knockdown of the p14ARF gene or p16INK4 gene present at the CDKN2A locus can improve the efficiency of direct reprogramming from human fibroblasts to neurons (Non-Patent Document 5). ).

Although p14ARF and p16INK4 are molecules that are present in different signaling pathways, both signaling pathways are involved in cell apoptosis and aging. Downstream of p14ARF in the first signaling pathway are MDM2, p53 and p21. In addition, cyclins D1, CDK4, CDK6, Rb and E2F are present downstream of p16INK4 in the second signal transduction pathway.

Human fibroblasts by knocking down the p53 gene located downstream of p14ARF in the first signaling pathway or by introducing a p53 dominant negative variant that inhibits the function of the p53 protein into human fibroblasts. It has been reported that the efficiency of reprogramming from cells to iPS cells can be improved (Non-Patent Document 6). It has also been reported that the efficiency of direct reprogramming from human fibroblasts to neurons can be improved by introducing a p53 dominant negative mutant into cells (Non-Patent Document 7).

Molofsky, A.V. et al., Genes & development, 2012, 26, 891-907 Krencik, R. et al., Nature biotechnology, 2011, 29, 528-534. Caiazzo, et al., Stem Cell Reports, 2015, 4, 25-36 Li, H. et al., Nature, 2009, 460, 1136-1139 Sun, C., et al., Nature Communications, 5, Article Number: 4112, June 17, 2014 Hong, H., et al., Nature, 2009, 460, 1132-1135 Liu, X., et al., Science China Life Sciences, 2014, 57, 867-875

When astrocytes are prepared by inducing differentiation from iPS cells, a period of several months is required, and the requirement for rapid supply of human astrocytes cannot be satisfied.

In addition, in the method reported in Non-Patent Document 3, only about 2% of astrocyte-like cells are contained in human neonatal fibroblasts even after introduction of NFIA, NFIB and SOX9 and culturing for 3 weeks. Not observed, its induction efficiency is extremely low.

Therefore, the present invention provides a novel method that enables rapid and highly efficient preparation of astrocyte-like cells having characteristics similar to those of human astrocytes, which are extremely difficult to obtain, using easily obtained human cells. Make it an issue.

As a result of diligent studies in view of the above problems, the present inventors have found that at least one of a specific inducer (NICD (Notch 1 intercellular domain)) and Tet2CD (TET (Ten-eleven translocation) 2 catalytic domain) in human somatic cells. And NFIA), it was found that human astrocyte-like cells can be prepared with high efficiency in an extremely short period of time without going through the preparation of iPS cells. Furthermore, we have introduced human astrocyte-like cells for an extremely short period of time even when the same inducer as above is introduced into pluripotent stem cells such as iPS cells and adult stem cells such as neural stem cells. It was found that it can be prepared with high efficiency. The present invention is based on these surprising findings.

That is, the present invention has the following aspects.
[1]
A method for preparing astrocyte-like cells from human cells in vitro, in the following steps:
It comprises (1) a step of introducing at least two kinds of inducers into human cells; and (2) a step of inducing differentiation of the human cells to obtain astrocyte-like cells.
Here, the inducing factor comprises NFIA and comprises at least one of NICD and Tet2CD.
The preparation method, wherein the human cell is a human somatic cell (excluding astrocytes), a human pluripotent stem cell, or a human adult stem cell.
[2]
The preparation method according to [1], wherein the inducing factor comprises NFIA, NICD and Tet2CD.
[3]
The preparation method according to [1] or [2], wherein the human cell is a human fibroblast.
[4]
The preparation method according to [1] or [2], wherein the human cell is a human iPS cell or a human neural stem cell.
[5]
The human cell is a cell derived from a patient with a nervous system disease, or a cell derived from a healthy person in which a mutation specifically present in the gene of the cell derived from the patient with a nervous system disease is artificially introduced into the gene. , [1] to [4].
[6]
The preparation method according to any one of [1] to [5], wherein the introduction of the inducing factor in the step (1) includes introducing at least one vector containing a nucleic acid encoding the inducing factor.
[7]
In the step (1)
Whether to inhibit the expression of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene.
Inhibits at least one selected from p14ARF, p53, p21, p16INK4a and Rb.
Increase the expression of at least one gene selected from the MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, or activate at least one selected from MDM2, cyclin D1, CDK4, CDK6 and E2F. The preparation method according to any one of [1] to [6], further comprising cyclinizing.
[8]
An astrocyte-like cell prepared by the method according to any one of [1] to [7].
[9]
A method for assessing the efficacy or toxicity of a test substance against nervous system diseases, the following steps:
(1) From cells derived from a patient with a nervous system disease or cells derived from a healthy person in which a mutation specifically present in the gene of a cell derived from the patient with a nervous system disease is artificially introduced into the gene, [1] to [ The step of preparing astrocyte-like cells by the method according to any one of 7]; and (2) The astrocyte-like cells obtained in step (1) are brought into contact with the test substance to bring the test substance into contact. The method comprising assessing efficacy or toxicity.
[10]
A method for assessing the efficacy or toxicity of a test substance against nervous system diseases, the following steps:
(1) A step of preparing astrocyte-like cells from cells derived from a healthy subject by the method according to any one of [1] to [7];
(2) In the astrocyte-like cells obtained in step (1), a step of knocking out or knocking down a gene related to a nervous system disease; and (3) the astrocyte-like cells obtained in step (2) and a test substance The method comprising contacting the test substance to evaluate the efficacy or toxicity of the test substance.
[11]
An in vitro neural tissue model comprising the astrocyte-like cells and neurons according to [8].
[12]
A method for assessing the efficacy or toxicity of a test substance against nervous system diseases, the following steps:
(1) From cells derived from a patient with a nervous system disease or cells derived from a healthy person in which a mutation specifically present in the gene of the cell derived from the patient with a nervous system disease is artificially introduced into the gene, [1] to [ A step of preparing astrocyte-like cells by the method according to any one of 7];
(2) A step of co-culturing the astrocyte-like cells obtained in step (1) and neurons to prepare a nerve tissue model;
(3) The method according to the above method, which comprises a step of contacting the nerve tissue model obtained in step (2) with a test substance to evaluate the efficacy or toxicity of the test substance.
[13]
A method for assessing the efficacy or toxicity of a test substance against nervous system diseases, the following steps:
(1) A step of preparing astrocyte-like cells from cells derived from a healthy subject by the method according to any one of [1] to [7];
(2) A step of knocking out or knocking down a gene related to a nervous system disease in the astrocyte-like cell obtained in the step (1);
(3) A step of co-culturing the astrocyte-like cells obtained in step (2) and neurons to prepare a nerve tissue model;
(4) The method according to the above method, which comprises a step of contacting the nerve tissue model obtained in step (3) with a test substance to evaluate the efficacy or toxicity of the test substance.
[14]
An inducer from human cells to astrocyte-like cells comprising at least two inducers or at least one vector containing a nucleic acid encoding the inducer as a component, wherein the inducer is NFIA. The inducer comprising, and containing at least one of NICD and Tet2CD, wherein the human cell is a human somatic cell (excluding astrocytes), a human pluripotent stem cell or a human adult stem cell.
[15]
Includes at least two inducers, or at least one vector containing a nucleic acid encoding the inducer; and an instruction manual describing the method according to any one of [1] to [7]. A kit for inducing human cells to astrocyte-like cells, wherein the inducer comprises NFIA and at least one of NICD and Tet2CD, wherein the human cell is a human somatic cell (astrocytosis). The kit, which is a human pluripotent stem cell or a human adult stem cell.
[16]
An expression inhibitor of at least one gene selected from p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene, and / or at least one inhibitor selected from p14ARF, p53, p21, p16INK4a and Rb, and / Or an expression enhancer of at least one gene selected from MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, and / or at least one selected from MDM2, cyclin D1, CDK4, CDK6 and E2F. The kit according to [15], further comprising an activator.

According to the method of the present invention, human astrocytes can be supplied in large quantities quickly. In addition, the human astrocytes obtained by the method of the present invention can be used to construct a novel in vitro disease model.

Induction after introduction of a lentiviral vector containing a nucleic acid encoding NFIA, a lentiviral vector containing a nucleic acid encoding NICD, and a lentiviral vector containing a nucleic acid encoding Tet2CD into human fibroblasts and culturing for 11 days. The percentage of GFAP-expressing cells produced is shown. The vertical axis shows the ratio of the number of GFAP positive cells to the number of DAPI-positive cells. In the figure, "Fibro" indicates human fibroblasts into which an inducing factor has not been introduced. "NFIA", "Tet2CD", and "NICD" indicate human fibroblasts into which NFIA, Tet2CD, and NICD have been introduced, respectively. In the figure, "NFIA / Tet2CD", "Tet2CD / NICD", and "NICD / NFIA" are NFIA and Tet2CD-introduced human fibroblasts, Tet2CD and NICD-introduced human fibroblasts, NICD and NFIA, respectively. Indicates human fibroblasts into which. In the figure, "NIFA / NICD / Tet2CD" indicates human fibroblasts into which NFIA, NICD and Tet2CD have been introduced. After co-culturing astrocyte-like cells with neurons, the image of the neuron by a fluorescence microscope and the measurement result of the length of the neurite extending from the neuron are shown. The scale bar in the image is 100 μm. Fluorescence microscopy images were obtained by labeling neurons with an anti-MAP2 antibody (Sigma). The vertical axis of the graph shows the average neurite length per neuron cell. Moreover, in the figure, "w / o" indicates the case where it is not co-cultured with astrocyte-like cells (that is, only neurons). In the figure, "w / NHDF-Ad" indicates a case where human adult skin fibroblasts (NHDF-Ad) and neurons are co-cultured. In the figure, "w / NHDF-Neo" indicates a case where human neonatal skin fibroblasts (NHDF-Neo) and neurons are co-cultured. In the figure, "w / iAs (NHDF-Ad)" indicates a case where astrocyte-like cells derived from human adult skin fibroblasts and neurons are co-cultured. In the figure, "w / iAs (NHDF-Neo)" indicates a case where astrocyte-like cells derived from human neonatal skin fibroblasts and neurons are co-cultured. In the figure, "w / hAstrocytes" indicates a case where human astrocytes and neurons are co-cultured. The measurement result of the glutamate uptake ability of astrocyte-like cells is shown. Human lt-NES cells (long-term self-renewing neuroepithelial stem cells) are a retrovirus vector containing a nucleic acid encoding NFIA, a retrovirus vector containing a nucleic acid encoding NICD, and a retrovirus vector containing a nucleic acid encoding Tet2CD. The percentage of induced GFAP-expressing cells after introduction into and culturing for 1 week and 2 weeks is shown. The vertical axis shows the ratio of the number of GFAP positive cells to the total number of cells. In the figure, "lt-NES iA" indicates astrocyte-like cells derived from lt-NES cells. Fluorescence microscopy images of astrocyte-like cells derived from human adult fibroblasts and lt-NES cells using a lentiviral vector containing all nucleic acids encoding NFIA, NICD, and Tet2CD are shown. It is shown that the expression level of CRYAB was increased in the in vitro model of Alexander disease obtained by inducing differentiation from lt-NES cells as compared with the control group. The vertical axis shows the ratio of the number of GFAP-positive and CRYAB (Alpha-crystallin B chain) -positive cells to the number of GFAP-positive cells. In the figure, "Control" indicates astrocyte-like cells derived from lt-NES cells into which a missense point mutation (C715T) has not been introduced, and "AxD" indicates an lt into which a missense point mutation (C715T) has been introduced. -Shows astrocyte-like cells derived from NES cells. When a lentivirus vector expressing short hairpin RNA (shRNA) for MeCP2 was introduced into astrocyte-like cells derived from fibroblasts and then co-cultured with mouse E15.5 primary cultured cerebral cortex neurons, the neurons were measured by fluorescence microscopy. The image and the measurement result of the length of the astrocyte extending from the neuron are shown. Fluorescence microscopy images were obtained by labeling neurons with an anti-MAP2 antibody (Sigma). In the figure, "w / iA (control)" shows the results of co-culture of astrocyte-like cells and neurons that do not express shRNA for MECP2 (control group). In the figure, "w / iA (shMeCP2)" shows the results of co-culture of astrocyte-like cells expressing shRNA for MECP2 and neurons. In the figure, "w / iA (Rescue)" indicates a co-culture of astrocyte-like cells and neurons expressing both shRNA for MECP2 and wild-type MECP2 whose expression is not suppressed by shRNA. The vertical axis of the graph shows the ratio of the average neurite length of the co-culture group to the average neurite length per neuron cell in the control group. Measurement of the length of neurites extending from neurons when a lentiviral vector expressing shRNA for MeCP2 was introduced into astrocyte-like cells derived from lt-NES cells and then cultured with mouse E15.5 primary cultured cerebral cortex neurons. The results are shown. The vertical axis shows the average neurite length per neuron cell. In the figure, "w / iA (shMeCP2)" shows the results of co-culture of astrocyte-like cells expressing shRNA for MECP2 and neurons. In the figure, "w / iA (Rescue)" indicates a co-culture of astrocyte-like cells and neurons expressing both shRNA for MECP2 and mutant MECP2 not suppressed by shRNA. The results when p16INK4a is introduced into cells in the induction of differentiation from human fibroblasts to astrocyte-like cells are shown. In the figure, "3 factors" are human fibroblasts into which a lentiviral vector containing a nucleic acid encoding NFIA, a lentiviral vector containing a nucleic acid encoding NICD, and a lentiviral vector containing a nucleic acid encoding Tet2CD have been introduced. Indicates that there is. In the figure, "+ p16INK4a" indicates the result when a lentiviral vector containing a nucleic acid encoding p16INK4a is introduced into cells in addition to the vector encoding the above three factors. In the figure, "+ Ctl" indicates the result when an empty lentiviral vector containing no gene sequence to be expressed was used instead of the lentiviral vector containing the nucleic acid encoding p16INK4a. The vertical axis of the bar graph on the left (% of GFAP (+) / DAPI) is the ratio of the number of GFAP-positive cells to the number of DAPI-positive cells (GFAP) after introducing the vector encoding the inducing factor into the cells and culturing for 11 days. The number of positive cells / the number of DAPI-positive cells) is shown in%. The vertical axis of the bar graph on the right (% of GFAP (+) / Initial cells) shows the number of GFAP-positive cells recovered after introducing the vector encoding the inducing factor into the cells and culturing for 11 days at the start of culturing. The ratio (number of GFAP-positive cells / number of initial cells) to the initial number of cells is shown in%. The results of knocking down the intracellular p16INK4a gene in inducing differentiation of human fibroblasts into astrocyte-like cells are shown. In the figure, the definitions of "3 factors", "% of GFAP (+) / DAPI" and "% of GFAP (+) / DAPI" are the same as in FIG. In the figure, "+ shp16INK4a" shows the result when a lentiviral vector containing a nucleic acid encoding short hairpin RNA (shp16INK4a) for the p16INK4a gene was introduced into cells in addition to the vector encoding the above three factors. In the figure, "+ Ctl" introduces into cells a lentiviral vector containing a nucleic acid encoding a short hairpin RNA targeting a sequence not present in humans, instead of a lentiviral vector containing a nucleic acid encoding shp16INK4a. The result when this is done is shown. The results of knocking down the intracellular p53 gene in the induction of differentiation from human fibroblasts to astrocyte-like cells are shown. In the figure, the definitions of "3 factors", "% of GFAP (+) / DAPI" and "% of GFAP (+) / DAPI" are the same as in FIG. In the figure, "+ shp53" shows the result when a lentiviral vector containing a nucleic acid encoding short hairpin RNA (shp53) for the p53 gene was introduced into cells in addition to the three factors. In the figure, "+ Ctl" introduces into cells a lentiviral vector containing a nucleic acid encoding a short hairpin RNA whose target sequence is a sequence not present in humans, instead of the lentiviral vector containing the nucleic acid encoding shp53. The result when this is done is shown. The results of knocking down the intracellular p14ARF gene in inducing differentiation of human fibroblasts into astrocyte-like cells are shown. In the figure, the definitions of "3 factors", "% of GFAP (+) / DAPI" and "% of GFAP (+) / DAPI" are the same as in FIG. In the figure, "+ shp14ARF" shows the result when a lentiviral vector containing a nucleic acid encoding short hairpin RNA (shp14ARF) for the p14ARF gene was introduced into cells in addition to the three factors. In the figure, "+ Ctl" means that instead of a vector containing a nucleic acid encoding shp14ARF, a lentiviral vector containing a nucleic acid encoding a short hairpin RNA targeting a sequence that does not exist in humans is introduced into cells. The result of is shown. The results of knocking down the intracellular p21 gene in the induction of differentiation from human fibroblasts to astrocyte-like cells are shown. In the figure, the definitions of "3 factors", "% of GFAP (+) / DAPI" and "% of GFAP (+) / DAPI" are the same as in FIG. In the figure, "+ shp21" shows the result when a lentiviral vector containing a nucleic acid encoding short hairpin RNA (shp21) for the p21 gene was introduced into cells in addition to the three factors. In the figure, "+ Ctl" means that instead of a vector containing a nucleic acid encoding shp21, a lentiviral vector containing a nucleic acid encoding a short hairpin RNA targeting a sequence that does not exist in humans is introduced into cells. The result of is shown. The percentage of induced GFAP-expressing cells after introducing one, two or three of mouse NICD, mouse Tet2CD, mouse NFIA, and mouse REST / NRSF-VP16 into mouse fibroblasts and culturing them. show. The vertical axis shows the number of GFAP-expressing cells per ^{1 cm 2.} The horizontal axis shows the introduced inducing factors.

One aspect of the present invention is a method of preparing astrocyte-like cells from human cells in vitro, wherein the following steps: (1) introducing at least two types of inducers into human cells; and (2) the above. It comprises the step of inducing differentiation of human cells to obtain astrocyte-like cells, wherein the inducing factor comprises NFIA and at least one of NICD and Tet2CD, wherein the human cell is a human somatic cell (astro). (Excluding astrocytes), the above-mentioned preparation method, which is a human pluripotent stem cell or a human adult stem cell. Hereinafter, the preparation method is also referred to as "the method for preparing astrocyte-like cells of the present invention" or "the method for preparing the astrocyte-like cells of the present invention".

As used herein, the term "astrocyte-like cell" is a cell obtained by inducing differentiation of human somatic cells (excluding astrocytes), human pluripotent stem cells, or human adult stem cells in vitro, and is astro. A cell that has similar properties to astrocytes. Specifically, it has the following characteristics.
(1) Expresses glial fibrous acidic protein (GFAP), which is a marker specific to astrocytes.
(2) It has the ability to support the growth of neurites from neurons.
(3) Has the ability to take up glutamic acid.
In the present specification, astrocyte-like cells are also referred to as "induced astrocyte cells" or "iA cells".

As used herein, the term "inducing factor" is a protein capable of inducing differentiation of human somatic cells (excluding astrocytes), human pluripotent stem cells and adult human stem cells into astrocyte-like cells. Inducing factors used in the method for preparing astrocyte-like cells of the present invention include NFIA (Nuclear factor I / A) and include at least one of NICD (Notch 1 intercellular domain) and Tet2CD (Tet2 catalytic domain). .. Specifically, the inducing factor may include NFIA and NICD, may include NFIA and Tet2CD, and may include NFIA, NICD and Tet2CD. Preferably, the inducing factors include NFIA, NICD and Tet2CD. In addition to the wild-type protein, the inducing factor has 90% or more, preferably 95% or more, more preferably 98% or more, particularly preferably 99% or more identity with the amino acid sequence of the wild-type protein, and is wild-type. It may be a mutant protein having the same ability to induce astrosite-like cells as the protein. For example, when mouse NFIA, mouse NICD and mouse Tet2CD are selected as inducers, not only the use of their wild-type proteins (SEQ ID NOs: 8, 9 and 10 respectively), but also 90% or more, preferably 95% or more of their sequences. The use of a mutant protein having an identity of% or more, more preferably 98% or more, particularly preferably 99% or more, and capable of inducing astrocyte-like cells is also the use of an inducing factor in the preparation method of the present invention. include. In addition to the inducer protein itself, the inducer may be in the form of a fusion protein of the protein and another protein, polypeptide, peptide or the like. The other protein, polypeptide or peptide is not particularly limited, but for example, a protein tag such as His tag, FLAG tag, Myc tag, V5 tag, PA tag and HA tag, and a cell membrane such as TAT peptide derived from HIV virus. Contains permeable peptides. As long as the fusion protein has the ability to induce into astrocyte-like cells, the use of the fusion protein is also included in the use of the inducer in the preparation method of the present invention.

The origin of the inducing factor used in the preparation method of the present invention is not particularly limited, but is preferably derived from a mammal, more preferably a primate (human, monkey, etc.), a rodent (mouse, rat, guinea pig, etc.). ), Cats, dogs, rabbits, sheep, pigs, cows, horses, donkeys, goats or ferrets, particularly preferably from mice or humans. The combination of inducers used in the preparation method of the present invention may be a combination of inducers derived from different species or a combination of inducers derived from the same species. Preferably, the combination of inducers used in the preparation method of the present invention is a combination of inducers derived from the same species.

As used herein, the term "somatic cell" refers to a cell in the germline (for example, an egg cell, a sperm cell, an egg cell, a sperm cell, etc. and their precursor cell) or a pluripotent undifferentiated cell derived from an early developmental embryo (for example). For example, it is a differentiated cell that constitutes an individual animal other than embryonic stem cells). The individual animal may be an adult, a foetation or an embryo. The somatic cell may be an established cell or a primary cultured cell isolated from a tissue, but it is preferable that the number of chromosomes is normal. The tissues and organs from which somatic cells are derived are not particularly limited, and include skin, liver, blood and the like. The properties of somatic cells are not particularly limited as long as they have lost even a part of the total differentiation potential of fertilized cells, and include, for example, fibroblasts, epithelial cells, hepatocytes, peripheral blood cells and the like.

In the method for preparing astrocyto-like cells of the present invention, the human somatic cells used as starting cells are not particularly limited as long as the astrosite-like cells are induced to differentiate, but are human fibroblasts or human peripheral blood cells. Is preferable.

As used herein, the term "pluripotent stem cell" includes all cells having pluripotency that can differentiate into all cells other than the placenta, and includes embryonic stem cells (ES cells) and induced pluripotent cells. Includes induced pluripotent stem cells (iPS cells).

An "ES cell" is a stem cell produced from an inner cell mass belonging to a part of an embryo in the blastocyst stage, which is the early stage of development of an animal.

"IPS cells" are cells that have been reprogrammed (reprogrammed) to have pluripotency similar to ES cells by introducing specific factors into mammalian somatic cells or undifferentiated stem cells. be.

iPS cells were first established by introducing the four factors Oct3 / 4, Sox2, Klf4, and c-Myc into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663- 676). Then, by introducing the same four factors into human fibroblasts, human iPS cells were also established (Takahashi K, Yamanaka S. et al., Cell, (2007) 131: 861-872.), And c-Myc was further added. We have also succeeded in establishing a method for establishing safer iPS cells with low induction of tumorigenesis, such as a method that does not include them (Nakagawa M, Yamanaka S., Nature Biotechnology, (2008) 26, 101-106).

In addition, artificial pluripotent stem cells produced by introducing factors different from the above (OCT3 / 4, SOX2, NANOG, LIN28) into human fibroblasts have been reported (Yu J., Thomson JA. et al., Science, 2007, 318: 1917-1920.). In addition, artificial pluripotent stem cells prepared by introducing 6 genes of OCT3 / 4, SOX2, KLF4, C-MYC, hTERT, and mSV40range T into skin cells have been reported (Park IH, Daley GQ. Et al., Nature). , 2007, 451: 141-146). In addition, induced pluripotent stem cells (Shi Y., Ding S. et al., Cell Stem Cell, 2008, Vol3, Issue 5,568) produced by introducing OCT3 / 4, KLF4, and low molecular weight compounds into mouse neural progenitor cells and the like. -574), induced pluripotent stem cells produced by introducing OCT3 / 4, KLF4 into mouse neural stem cells endogenously expressing SOX2, C-MYC (Kim JB., Scholer HR. Et al., Nature, Nature, (2008) 454,646-650), induced pluripotent stem cells produced using Dnmt inhibitors and HDAC inhibitors without using C-MYC (Huangfu D., Melton, DA. Et al., Nature Biotechnology, (2008) 2008) 26, No7,795-797) has been reported.

In the present specification, "induced pluripotent stem cells (iPS cells)" are known artificial pluripotent stem cells and artificial pluripotency equivalent thereto, as long as they satisfy the above definition and do not impair the object of the present invention. It includes all stem cells, and the cell source, introduction factor, introduction method, etc. are not particularly limited.

As used herein, an "adult stem cell" is an undifferentiated cell found in a differentiated tissue and has the ability to proliferate and differentiate into one or more cell types. Examples of adult stem cells include neural stem cells, hematopoietic stem cells, mesenchymal stem cells and the like. Neural stem cells are preferable as the adult stem cells used in the method for preparing astrocyte-like cells of the present invention. The origin of adult stem cells used in the method for preparing astrocyte-like cells of the present invention is not particularly limited, and may be obtained from a human living body or derived from pluripotent stem cells. It may be an astrocyte. As the adult stem cells used in the method for preparing astrocyte-like cells of the present invention, adult stem cells derived from pluripotent stem cells are preferable, and human nerve stem cells derived from human iPS cells (for example, derived from human iPS cells). Lt-NES cells) are more preferred.

In one embodiment of the method for preparing astrocyte-like cells of the present invention, the starting cell, the human cell, may be a cell derived from a patient with a nervous system disease. The nervous system disease is not particularly limited, and examples thereof include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, autism, Alexander disease, and Rett syndrome.

In one embodiment of the method for preparing astrocyte-like cells of the present invention, the starting cell, the human cell, may be a cell derived from a healthy person who does not suffer from a nervous system disease. The cell derived from a healthy person may be a cell derived from a healthy person into which a mutation specifically present in the gene of a cell derived from a patient with a nervous system disease has been introduced into the gene. Mutations can be introduced by genome editing methods using methods well known to those skilled in the art (eg, CRISPR-Cas9 system (Ran, FA et al., Cell, 2013, 154, 1380-1389), genetic modification by ZFN (Urnov, F. et al.) , Nature, 2005, 435, 646-651), or genetically modified by TALEN (Mahfouz, M et al., PNAS, 2011, 108, 2623-2628)).

In the method for preparing astrocyte-like cells of the present invention, the method for introducing an inducing factor into a human cell as a starting cell is not particularly limited, and for example, the inducing factor itself may be directly introduced into a cell (protein introduction method). Alternatively, at least one vector containing a nucleic acid encoding an inducing factor may be introduced into human cells to express the inducing factor (gene transfer method).

The method for introducing the inducer into human cells by introducing the protein itself is not particularly limited, and any method well known to those skilled in the art may be used. For example, a method using a protein transfer reagent, a method using a protein transfer domain (PTD) or a cell-penetrating peptide (CPP) fusion protein, a microinjection method, an electroporation method and the like can be mentioned.

On the other hand, when a gene transfer method for introducing an inducing factor into human cells is used, a method well known to those skilled in the art is first used, downstream of an appropriate promoter for expressing the nucleic acid encoding the inducing factor in human cells. Make at least one inserted vector. As used herein, the "nucleic acid encoding an inducing factor" may be DNA, RNA, or a DNA / RNA chimera. Further, the nucleic acid may be double-stranded or single-stranded. Preferably, the nucleic acid is double-stranded DNA, especially cDNA. The nucleic acid encoding the inducing factor may be a mutant sequence thereof as long as the expressed protein has the same ability to induce astrocyte-like cells as the wild-type inducing factor. In the present specification, "at least one vector containing a nucleic acid encoding an inducing factor" may be incorporated into each of the nucleic acids on a separate vector, or two or more types, preferably two or three types, may be incorporated into one vector. Indicates that the nucleic acid of the above may be incorporated.

The vector that can be used in the method for preparing astrosite-like cells of the present invention is not particularly limited, and is, for example, a plasmid vector, a virus vector (for example, a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a Sendai virus vector). Examples include artificial chromosome vectors and episomal vectors. As the vector used in the method for preparing astrocyte-like cells of the present invention, a lentiviral vector is preferable.

As a method for introducing the vector into human cells, any method well known to those skilled in the art, such as an electroporation method, a calcium phosphate method, a lipofection method, or a method via virus infection, may be used. In this way, the inducing factor can be introduced into human cells by introducing a vector capable of expressing the inducing factor into human cells and expressing the inducing factor in the human cells.

By culturing human cells into which an inducing factor has been introduced, differentiation can be induced into astrocyte-like cells. The medium used is not particularly limited, but for example, Dulbecco's modified Eagle's medium (DMEM) and astrocyte growth medium (AGM medium ™, Lonza) can be used. The culture temperature is preferably 33 to 37 ° C, more preferably 37 ° C. The culture period is not particularly limited as long as the astrocyte-like cells can be recovered, but usually, the astrocyte-like cells can be recovered in about 1 to 2 weeks.

Signal transduction pathways mediated by p14ARF, MDM2, p53 and p21 exist as signaling pathways involved in cell apoptosis and aging. Specifically, p14ARF inhibits MDM2. In addition, MDM2 inhibits the activation of p53 by inhibiting transcriptional regulation by p53, promoting the degradation of p53, and promoting the nuclear transport of p53. In addition, p53 activates p21 by transcriptional activation of the p21 gene. In addition, p21 promotes cell apoptosis and aging by regulating the expression of genes associated with apoptosis and aging.

In addition, as another signal transduction pathway involved in cell apoptosis and aging, there is a signal transduction pathway mediated by p16INK4a, cyclin D1, CDK4, CDK6, Rb and E2F. Specifically, p16INK4a inhibits the activation of cyclins D1, CDK4 and CDK6 by inhibiting the binding of cyclin D1 to CDK4 / 6. In addition, the complex of cyclin D1 and CDK4 / 6 inhibits the binding between Rb and E2F by phosphorylating Rb. Since E2F is suppressed by the binding of Rb protein, it has a transcriptional activation effect by phosphorylation of Rb. E2F suppresses cell apoptosis and aging by regulating the expression of genes associated with apoptosis and aging.

It has been reported that inhibition of signal transduction originating from p14ARF or p16INK4a in the above two pathways increases the efficiency of induction into iPS cells or neurons. Surprisingly, it was revealed that inhibition of the signal transduction also increases the efficiency of induction into astrocyte-like cells.

Therefore, in step (1) of the method for preparing astrosite-like cells of the present invention, the expression of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene is inhibited, or p14ARF, Inhibit at least one selected from p53, p21, p16INK4a and Rb, or increase the expression of at least one gene selected from the MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, or It may further include activating at least one selected from MDM2, cyclin D1, CDK4, CDK6 and E2F. Preferably, in step (1) of the method for preparing astrosite-like cells of the present invention, the expression of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene is inhibited or p14ARF is inhibited. , P53, p21, p16INK4a and Rb, or increase the expression of at least one gene selected from the MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene. Further may be included. More preferably, in the step (1) of the method for preparing astrosite-like cells of the present invention, the expression of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene is inhibited. It may further include inhibiting at least one selected from p14ARF, p53, p21, p16INK4a and Rb. More preferably, in the step (1) of the method for preparing astrosite-like cells of the present invention, the expression of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene is inhibited. Further may be included. Particularly preferably, in the step (1) of the method for preparing astrosite-like cells of the present invention, further including inhibiting the expression of at least one gene selected from the p14ARF gene, p53 gene, p21 gene and p16INK4a gene. good.

The method for inhibiting the expression of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene is not particularly limited, and can be carried out by any method well known to those skilled in the art. For example, it may be carried out by introducing nucleic acids (eg, RNAi-causing ones such as siRNA, shRNA and miRNA) into human cells. In addition, knockout by genome editing using the CRISPR-Cas9 system (Ran, FA et al., Cell, 2013, 154, 1380-1389) and gene modification using ZFN (Urnov, F. et al., Nature, 2005, 435, 646-651). ), Or by genetic modification using TALEN (Mahfouz, M et al., PNAS, 2011, 108, 2623-2628)). Inhibition of the expression of the gene is preferably carried out using siRNA, shRNA or miRNA that knocks down the target gene, and more preferably using shRNA.

When inhibition of the expression of the above gene is performed using shRNA, it is not particularly limited, but is preferably encoded by DNA having the base sequence shown by SEQ ID NO: 14 or DNA having the base sequence shown by SEQ ID NO: 15. RNA (shRNA that knocks down the p14ARF gene), RNA encoded by the DNA having the base sequence shown by SEQ ID NO: 16 or the DNA having the base sequence shown by SEQ ID NO: 17 (shRNA that knocks down the p53 gene gene), It has the RNA encoded by the DNA having the base sequence shown by SEQ ID NO: 18 or the DNA having the base sequence shown by SEQ ID NO: 19 (shRNA that knocks down the p21 gene), or the base sequence shown by SEQ ID NO: 20. It is performed using RNA encoded by DNA or DNA having the base sequence shown by SEQ ID NO: 21 (shRNA that knocks down the p16INK4a gene).

The method for inhibiting at least one selected from p14ARF, p53, p21, p16INK4a and Rb is not particularly limited and can be carried out by any method well known to those skilled in the art. For example, it may be carried out by introducing a p53 dominant negative mutant that inhibits p53, an inactive Rb mutant, pifithrin-α (a transcription inhibitor of p53), or the like into human cells.

The method for increasing the expression of at least one gene selected from the MDM2 gene, the cyclin D1 gene, the CDK4 gene, the CDK6 gene and the E2F gene is not particularly limited, and can be carried out by any method well known to those skilled in the art. For example, an expression vector incorporating the above genes can be used to force expression of these genes in human cells, or an inactive ZFN nuclease fused with a transcriptional activator such as VP16, VP64, or VP128 (Sanchez, Sanchez, JP. Et al., Plant Biotechnol J, 2006, 4 (1), 103-114), Inactive TALEN nuclease (Perez-Pinera, P. et al., Nat Methods, 2013, 10, 239-242) or Inactive Cas9 It may be carried out by utilizing a nuclease (Konermann, S. et al., Nature, 2015, 517, 583-588) to increase the endogenous transcript of the gene.

The method for activating at least one selected from MDM2, cyclin D1, CDK4, CDK6 and E2F is not particularly limited, and can be performed by any method well known to those skilled in the art.

Inhibition and increase of the expression of the above gene, and inhibition and activation of the gene product of the gene may be carried out at the same time as the introduction of the inducing factor into human cells, or may be carried out before or after the introduction of the inducer.

One aspect of the present invention relates to astrocyte-like cells prepared by the preparation method of the present invention. One embodiment of the present invention comprises the following steps: (1) introducing at least two types of inducers into human cells; and (2) inducing differentiation of the human cells to obtain astrocyte-like cells. Astrocyte-like cells prepared by the method comprising, wherein the inducer comprises NFIA and at least one of NICD and Tet2CD, wherein the human cell is a human somatic cell (excluding astrocytes). ), The astrocyte-like cell, which is a human pluripotent stem cell or a human adult stem cell.

For example, when cells derived from a patient with a nervous system disease are used as starting cells in the method for preparing astrocyte-like cells of the present invention, or mutations specifically present in cells derived from a patient with a nervous system disease are artificially introduced. When the cells of healthy individuals are used, the prepared astrocyte-like cells retain the genetic characteristics specific to the nervous system disease. Therefore, the astrocyte-like cells can be used as a model of a nervous system disease for elucidating the cause of the onset of the nervous system disease and the mechanism of pathological progression. In addition, the astrocyte-like cells can be used to evaluate the efficacy or toxicity of the test substance against the nervous system disease, and can be used for drug discovery research.

One aspect of the present invention is a method for evaluating the efficacy or toxicity of a test substance against a nervous system disease, wherein the following steps: (1) cells derived from a nervous system disease patient or cells derived from a nervous system disease patient. A step of preparing astrocyte-like cells by the method for preparing astrocyte-like cells of the present invention from cells derived from a healthy person into which a mutation specifically present in is introduced; and (2) with the astrocyte-like cells. The method relates to the method comprising contacting with a test substance to evaluate the efficacy or toxicity of the test substance. The nervous system disease is not particularly limited, and examples thereof include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, autism, Alexander disease, and Rett syndrome.

Astrocyte-like cells prepared from cells derived from healthy subjects by the preparation method of the present invention may be knocked out or knocked down by genes related to nervous system diseases. As used herein, the term "nervous system disease-related gene" means that the gene does not function normally due to mutation or the like, and it is clear or may cause nervous system disease directly or indirectly. Is a gene. Knockouts or knockouts are knockouts by genome editing using methods well known to those skilled in the art (eg, CRISPR-Cas9 system (Ran, FA et al., Cell, 2013, 154, 1380-1389), and genetic modification using ZFNs (Urnov, Urnov, Knockout by F. et al., Nature, 2005, 435, 646-651), knockout by gene modification using TALEN (Mahfouz, M et al., PNAS, 2011, 108, 2623-2628), and siRNA, shRNA, miRNA, etc. It can be done by knocking down with the RNAi used). The obtained astrocyte-like cells can be used as a model for nervous system diseases.

One aspect of the present invention is a method for evaluating the efficacy or toxicity of a test substance for nervous system diseases, and the following steps: (1) A method for preparing astrocyte-like cells of the present invention from cells derived from healthy subjects. To prepare astrocyte-like cells; (2) In the astrocyte-like cells obtained in step (1), knock out or knock down a gene related to a neurological system disease; and in (3) step (2). The present invention relates to the above method, which comprises contacting the obtained astrocyte-like cells with a test substance to evaluate the efficacy or toxicity of the test substance. The nervous system disease is not particularly limited, and examples thereof include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, autism, Alexander disease, and Rett syndrome.

One aspect of the invention relates to an in vitro neural tissue model comprising astrocyte-like cells and neurons prepared by the preparation method of the present invention. Hereinafter, the nerve tissue model is also referred to as a “nerve tissue model of the present invention”.

The astrosite-like cells used in the nervous tissue model of the present invention may be cells derived from cells derived from a neurological disease patient or cells derived from healthy subjects. Or, it may be a cell derived from a healthy person in which a mutation specifically present in the gene of a cell derived from a patient with a nervous system disease is artificially introduced into the gene.

Further, the astrocyte-like cells used in the neural tissue model of the present invention are cells derived from healthy subjects and induced to differentiate, even if the neural disease-related genes are knocked out or knocked down. good.

The neuron used in the nervous tissue model of the present invention is not particularly limited as long as it is a mammalian-derived neuron, and is, for example, a mouse neuron or a human neuron. In addition, the neuron may be a neuron derived from a patient with a nervous system disease or a neuron derived from a healthy person, and a mutation specifically present in the gene of a cell derived from a patient with a nervous system disease is introduced into the gene. It may be a neuron derived from a healthy person. The neuron may be a pluripotent stem cell derived from a patient with a nervous system disease or a neuron induced to differentiate from an adult stem cell, or may be a pluripotent stem cell derived from a healthy person or a neuron induced to differentiate from an adult stem cell. good.

The neural tissue model of the present invention is obtained by co-culturing astrocyte-like cells and neurons prepared by the preparation method of the present invention. The medium used in the co-culture is not particularly limited, but a medium in which nerve cells can survive is desirable. For example, Neurobasal (registered trademark) medium (Life Technologies), N2 supplement (Thermo Fisher Scientific) and B27 (registered trademark) Supplement with Gibco (registered trademark) B27 (registered trademark) Supplement (Thermo Fisher Scientific) added. The added DMEM / F12 medium can be used. The culture temperature is preferably 33 to 37 ° C, more preferably 37 ° C. The culture period is not particularly limited, but the analysis can usually be performed in about 1 to 2 weeks.

One aspect of the present invention is a method of preparing a nervous tissue model, in which the following steps: (1) a step of introducing at least two types of astrocytes into human cells; (2) inducing differentiation of the human cells. A step of obtaining astrocyte-like cells; and (3) a step of co-culturing the astrocyte-like cells obtained in step (2) with neurons, wherein the inducing factor contains NFIA and The present invention relates to the preparation method comprising at least one of NICD and Tet2CD, wherein the human cell is a human somatic cell (excluding astrocytes), a human pluripotent stem cell or a human adult stem cell.

One aspect of the present invention is a method for evaluating the efficacy or toxicity of a test substance against a nervous system disease, wherein the following steps: (1) cells derived from a nervous system disease patient or cells derived from the nervous system disease patient. Step of preparing astrosite-like cells by the method for preparing astrocyto-like cells of the present invention from cells derived from a healthy person in which a mutation specifically present in the gene of is artificially introduced into the gene; (2) A step of co-culturing the astrosite-like cells obtained in (1) and neurons to prepare a neural tissue model; (3) Contacting the neural tissue model obtained in step (2) with a test substance , The method comprising assessing the efficacy or toxicity of the test substance. In addition, one aspect of the present invention is a method for evaluating the efficacy or toxicity of a test substance for nervous system diseases, and the following steps: (1) From cells derived from healthy subjects to the astrocyte-like cells of the present invention. Steps of preparing astrocyte-like cells by the preparation method; (2) Steps of knocking out or knocking down nervous system disease-related genes in the astrocyte-like cells obtained in step (1); (3) Steps (2) Steps of co-culturing the astrocyte-like cells and neurons obtained in the above step to prepare a neural tissue model; (4) The neural tissue model obtained in step (3) is brought into contact with the test substance to obtain the test substance. The method comprises the steps of assessing the efficacy or toxicity of. Nervous system diseases include, but are not limited to, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, autism, Alexander disease, Rett syndrome and the like.

One aspect of the present invention is an inducer from human cells to astrocyte-like cells, which comprises at least two kinds of inducers or at least one vector containing a nucleic acid encoding the inducer as a component. , The inducer comprises NFIA and at least one of NICD and Tet2CD, wherein the human cell is a human somatic cell (excluding astrocytes), a human pluripotent stem cell or a human adult stem cell. With respect to the agent (hereinafter, also referred to as "inducing agent of the present invention"). The inducer of the present invention can be used in the method for preparing astrocyte-like cells of the present invention. For example, the inducer is brought into contact with a human cell, an inducer, or at least one vector containing an inducer-encoding nucleic acid is introduced into the cell, and then the cell is cultured under appropriate conditions to induce the cell into astrocytes. It can induce differentiation into astrocytes.

One aspect of the invention includes at least two inducers, or at least one vector containing a nucleic acid encoding the inducer; and an instruction manual describing the method for preparing astrocyte-like cells of the present invention. , A kit for inducing human cells to astrocyte-like cells, wherein the inducer comprises NFIA and at least one of NICD and Tet2CD, wherein the human cell is a human somatic cell (astrocyte). (Excluding), the kit is a human pluripotent stem cell or a human adult stem cell (hereinafter, also referred to as “kit of the present invention”). The instruction manual in the kit of the present invention may be provided on the web without being included in the kit of the present invention.

The kit of the present invention is selected from expression inhibitors of at least one gene selected from p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene, and / or p14ARF, p53, p21, p16INK4a and Rb. At least one inhibitor and / or an expression enhancer of at least one gene selected from the MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, and / or MDM2, cyclin D1, CDK4, CDK6. And at least one activator selected from E2F may be further included. Preferably, an expression inhibitor of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene, and / or at least one selected from p14ARF, p53, p21, p16INK4a and Rb. Inhibitors and / or expression enhancers for at least one gene selected from the MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene may be further included. More preferably, an expression inhibitor of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene, and / or at least one selected from p14ARF, p53, p21, p16INK4a and Rb. It may further contain one inhibitor. More preferably, it may further contain an expression inhibitor of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene. Particularly preferably, it may further contain an expression inhibitor of at least one gene selected from the p14ARF gene, p53 gene, p21 gene and p16INK4a gene.

The expression inhibitor of the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene and the inhibitor of the gene product of these genes are not particularly limited, but can be used to inhibit the expression of the gene. Used to inhibit those listed in (eg, siRNA, shRNA (eg, RNA encoded by DNA having the base sequences set forth in SEQ ID NOs: 14-21) and nucleic acids such as miRNA), and the gene product thereof. The ones listed above (eg, p53 dominant negative variants, inactive Rb variants, and piffithrin-α) can be used as possible.

The expression enhancer for the MDM2 gene, the cyclone D1 gene, the CDK4 gene, the CDK6 gene and the E2F gene, and the activator of the gene product of these genes are not particularly limited, but can be used to increase the expression of the gene. Those listed above (eg, expression vectors for these genes, inactive TALEN nucleases, inactive TALEN nucleases and inactive Cas9 nucleases), and those capable of activating the gene product. Those well known to those skilled in the art can be used.

The present invention will be described in more detail with reference to the following examples and reference examples, but the present invention is not limited thereto.

Example 1 Induction of differentiation of human fibroblasts into astrosite-like cells (1) Preparation of lentiviral vector containing nucleic acid encoding inducing factor NICD, lentiviral vector containing nucleic acid encoding NFIA (SEQ ID NO: 1) A lentiviral vector containing the encoding nucleic acid (SEQ ID NO: 2) and a lentiviral vector containing the nucleic acid encoding Tet2CD (SEQ ID NO: 3) were prepared. Specifically, lentivirus plasmids expressing inducing factors (NFIA, NICD or Tet2CD) downstream of the CBh promoter were obtained from pCAG-HIVgp and pCMV-VSV-G-RSV-Rev plasmids (obtained from Dr. Hiroyuki Miyoshi, RIKEN). At the same time, it was introduced into 293T cells in Gibco (registered trademark) FreeStyle (trademark) 293 Expression Medium (manufactured by Invitrogen) (hereinafter, also simply referred to as "Freestyle (trademark)"). After 16-20 hours of introduction, the medium was replaced with fresh medium. After further culturing for 48 hours, the culture supernatant was filtered using a 0.45 μm filter. The obtained supernatant was used as a stock solution or concentrated, and then introduced into various cells as a lentiviral vector.

(2) Introduction of lentiviral vector into human fibroblasts and induction of differentiation DMEM medium containing human adult fibroblasts (NHDF-Ad, Lonza) was placed in a 6-well plate or a 10 cm dish in 5 × 10 ^{4 respectively.} Seeded at a cell / well or 3 × 10 ⁵ cell / dish density. Twenty-four hours after sowing, the medium was replaced with a medium containing the lentiviral vector prepared in (1) and 4 μg / mL polybrene. After 16-20 hours of infection, the medium was replaced with fresh medium. The cells were maintained in medium for an additional 48 hours and then grown using AGM medium ™ (Lonza). The medium was changed every 3 days until analysis. Analysis was performed 2-3 weeks after infection.

(3) Analysis by immunocytochemistry
The obtained astrocyte-like cells were analyzed by immunocytochemistry according to the method described in Kohyama, J. et al., The Journal of cell biology, 2010, 189, 159-170. Specifically, cells were fixed with 4% paraformaldehyde at room temperature. After fixation, wash the cells with phosphate buffered saline (PBS), and then rinse the cells with 0.3% Triton / 5% FBS / PBS solution (antibody dilution solution) containing primary antibody at room temperature for 3 hours or 4 degrees. Treated in the evening. After the primary antibody treatment, the cells were washed with PBS, treated with an antibody diluted solution containing a secondary antibody bound with a fluorescent marker for 60 to 90 minutes at room temperature, and washed with PBS. Nuclei were stained with DAPI or Hoechst. Stained cells were mounted on glass slides.

(4) Results When NFIA was introduced into human fibroblasts, no GFAP-expressing cells were observed. When mouse neurofibroblasts were used, differentiation could be induced into GFAP-expressing cells by introducing only NFIA (see Reference Example 1 below).
GFAP is expressed when NFIA and NICD are introduced into human fibroblasts, when NFIA and Tet2CD are introduced into human fibroblasts, and when NFIA, NICD and Tet2CD are introduced into human fibroblasts. It was possible to induce differentiation into astrocyte-like cells. In particular, when three inducing factors were introduced, differentiation into astrocyte-like cells could be induced with high efficiency of 45.1% (Fig. 1).
It was confirmed that the obtained astrocyte-like cells did not express smooth muscle actin (SMA), which is normally expressed in fibroblasts.

Example 2
Quantification of neurite length from neurons when astrocyte-like cells and neurons are co-cultured A functional feature of astrocytes is the ability to support the growth of neurites from neurons. In order to clarify that the astrosite-like cells obtained in Example 1 have this function, human neonatal skin fibroblasts (NHDF-Neo) and human adult skin fibroblasts (NHDF-Ad) (both). A lentiviral vector incorporating NFIA cDNA (SEQ ID NO: 1) and a lentivirus incorporating NICD cDNA (SEQ ID NO: 2) in the same manner as in Example 1 using (purchased from Lonza) as a starting cell. Millicell cell culture inserts (24 holes, PCF membrane, 0.4 μm) were introduced into the vector and the lentiviral vector incorporating the Tet2CD cDNA (SEQ ID NO: 3) and cultured for 2 to 3 weeks to induce differentiation. , Purchased from Millipore) and cultured at 37 ° C for one day. In addition, E15.5 mouse-derived cerebral cortical neurons were seeded on glass coverslips using DMEM / F12 medium supplemented with Gibco® B27® Supplement and cultured at 37 ° C. for one day. After replacing the medium in the Millicell cell culture insert seeded with astrocyte-like cells with B27 / DMEM / F12 medium, the Millicell cell culture insert was set on the coverslip for 3 days and co-cultured at 37 ° C. As control groups, human neonatal fibroblasts (NHDF-Neo), human adult skin fibroblasts (NHDF-Ad), and human primary cultured astrocytes (HNA) were co-cultured with cerebral cortical neurons derived from E15.5 mice. Was used. To quantify the length of neurites, after labeling neurons with an anti-MAP2 antibody (Sigma), images were acquired with a fluorescence microscope, and Image J software (Meijering et al., Journal) having a neuron J plug-in was obtained. Of the International Society for Analytical Cytology, 2004, 58, 167-176). The results are shown in FIG. When the astrocyte-like cells and neurons were co-cultured, the promotion of neurite outgrowth from the neurons was significantly observed as compared with the case where the control group, fibroblasts and neurons, were co-cultured.

Example 3
Measurement of glutamate uptake ability of astrocyte-like cells A functional feature of astrocytes is the ability to take up glutamate. In order to clarify that the astrocyte-like cells obtained in Example 1 have this function, the following experiments were carried out. Human adult skin fibroblasts (NHDF-Ad), astrocyte-like cells obtained by introducing NFIA, NICD and Tet2CD in Example 1 and human primary cultured astrocytes are coated with poly-L-Lysine (Sigma). The seeds were sown on a 24-well plate. Sodium ion-containing buffer or sodium to which various cells were added with L-glutamic acid (Nacalai) and tritium-labeled L-glutamic acid (L- [2,3,4-3H]) (American Radiolabeled Chemicals Inc.). Incubation was carried out at 37 ° C. using ion-free buffer. After incubation, cells were washed with sodium ion-free buffer and lysed with 0.1N NaOH solution. Cytolysis was mixed with a liquid scintillation cocktail (National Diagnostics) and the radiation dose was measured using a liquid scintillation counter. In addition, the total amount of protein in various cells was quantified by the BCA method. The amount of glutamic acid uptake was evaluated by correcting the amount of sodium ion-dependent uptake with the total amount of protein. The results are shown in FIG.

Example 4 Induction of differentiation of human lt-NES cells into astrocyte-like cells Neural stem cells induced from human ES cells and iPS cells are known to resist differentiation into astrocyte-like cells (Falk, A. et al., PloS ONE, 2012, Vol. 7 (1), e29597). The effect of introducing the inducer used in the present invention was tested on such neural stem cells.

As starting cells, lt-NES cells derived from human iPS cells (obtained by Dr. Austin Smith, University of Cambridge) were used. lt-NES cells maintain stable proliferative capacity and properties as early neuroepithelial cells in the developing brain. lt-NES cells are known to have weaker glial formation properties (Falk, A. et al., PloS ONE, 2012, Vol. 7 (1), e29597).

A retrovirus vector was prepared as follows using a plasmid vector incorporating NFIA cDNA, a plasmid vector incorporating NICD cDNA, and a plasmid vector incorporating Tet2CD cDNA (obtained from Cell Biolabs, Inc). ..
The plasmid vector was introduced into Plat-E cells in FreeStyle ™. After 16-20 hours of introduction, the medium was replaced with fresh medium. After further culturing for 48 hours, the culture supernatant was filtered using a 0.45 μm filter. The obtained supernatant was used as a retrovirus vector in this example after being undiluted or concentrated.
FreeStyle with lentiviral plasmids expressing mouse Slc7a1 downstream of the UbC promoter (Takahashi, K. et al., Cell, 2007, 131 (5): 861-72.), PCAG-HIVgp and pCMV-VSV-G-RSV-Rev plasmids. It was introduced into 293T cells in ™, and the prepared lentivirus was introduced into lt-NES cells.
Then, the retrovirus vector prepared above is placed in a medium containing the lt-NES cells (DMEM / F12 medium containing 1% N2 Supplement, 0.1% B27 Supplement (hereinafter, also referred to as “liter-NES medium”)). In addition, the cells were cultured at 37 ° C. for 24 hours, and then the medium was exchanged for lt-NES medium containing 1% FBS to prepare astrocyte-like cells expressing GFAP. One week after the introduction of the retrovirus vector, the proportion of GFAP-expressing cells was more than 20% of the total. Furthermore, after 2 weeks, almost all lt-NES cells were induced into GFAP-expressing cells (Fig. 4).

Example 5 Induction of differentiation of human iPS cells into astrosite-like cells A culture vessel in which human iPS cells (obtained from the Center for iPS Cell Research and Application, Kyoto University) were coated with iMatrix-511 (purchased from Nippi Co., Ltd.), and AK03. Maintenance culture was performed using a medium (purchased from Ajinomoto Co., Ltd.). In the same manner as in Example 1 (1), a lentiviral vector containing a nucleic acid encoding an inducing factor was prepared and introduced into human iPS cells. The iPS cells into which the lentiviral vector had been introduced were subcultured to an iMatrix-511 uncoated culture vessel using AGM medium 72 hours after the introduction. The medium was changed every 3 to 4 days to prepare astrocyte-like cells expressing GFAP from human iPS cells. As the inducing factor, three types of NFIA, NICD and Tet2CD were used. Two weeks after the introduction of the vector, the proportion of GFAP-expressing cells was about 80% of the total.

Example 6 Preparation of astrocyte-like cells using a viral vector containing all nucleic acids encoding NFIA, NICD and Tet2CD, or an episomal vector (1) Tet2CD, NFIA and NICD cDNAs are porcine teschovirus-1 derived 2A peptides. It was ligated using cDNA. A lentiviral vector incorporating this sequence was introduced into adult human fibroblasts (NHDF-Ad, Lonza) and cultured for 11 days. The cells obtained were GFAP positive. A fluorescence micrograph is shown in FIG.
(2) Tet2CD, NFIA, and NICD cDNAs were ligated using 2A peptide cDNA derived from porcine teschovirus-1, and an episomal vector incorporating this sequence was prepared.
Then, the episomal vector prepared above was introduced into the lt-NES cells, and the cells were cultured for 7 days using a medium for lt-NES containing 1% FBS. Then, the cells were cultured in AGM medium for another 7 days. The cells obtained were GFAP positive. A fluorescence micrograph is shown in FIG.

Example 7 Preparation of an in vitro disease model of Alexander disease Alexander disease is known to be a disease caused by a mutation in the GFAP gene (Brenner, M. et al., Nature genetics, 2001, 27, 117-120). In the GFAP gene of Alexander disease patients, arginine 239 of GFAP is mutated to cysteine (R239C) due to a missense point mutation (C715T) in the gene. In addition, it is known that the expression of αB crystallin (CRYAB) is enhanced in astrocytes of Alexander disease patients (Der Perng, M. et al., American journal of human genetics, 2006, 79, 197-213). It is also known that mutant GFAP proteins aggregate to produce Rosenthal fibrils in astrocytes of Alexander disease patients (Dinda, AK et al., Acta neuropathologica, 1990, 79, 456-460).

In an in vitro disease model of Alexander disease, it was tested whether the astrocyte-like cells of the present invention could be used.
Using the CRISPR-Cas9 system (Ran, FA et al., Cell, 2013, 154, 1380-1389), lt-NES cells having a missense point mutation (C715T) in the GFAP gene were prepared. Then, in the same manner as in Example 4, a retroviral vector containing a nucleic acid encoding NFIA, a retroviral vector containing a nucleic acid encoding NICD, and a retroviral vector containing a nucleic acid encoding Tet2CD were introduced into the cells. Astrosite-like cells expressing GFAP were prepared.

In the obtained astrocyte-like cells, significantly higher expression of CRYAB was observed as compared with the control group (astrocyte-like cells derived from lt-NES cells before introducing a missense point mutation (C715T)). (Fig. 6). Furthermore, Rosenthal fibers were observed in the obtained astrocyte-like cells. The obtained astrocyte-like cells can be used as an in vitro disease model of Alexander disease for elucidation of the molecular mechanism and pathogenesis of the disease and screening of a therapeutic drug for the disease.

Example 8 Creating an In vitro Disease Model for Rett Syndrome Rett Syndrome is caused by a mutation in the MECP2 gene located on the X chromosome. Recent findings on mouse genes suggest that astrocytes play an important role in the development of Rett syndrome (Ballas, N et al., Nature neuroscience, 2009, 12, 311-317; Nguyen, MV et al., The Journal of neuroscience, 2012, 32, 10021-10034). In addition, astrocyte-like cells derived from iPS cells derived from patients with Rett syndrome have been reported to adversely affect neuronal maturation (Williams et al., Hum Mol Genet. 2014 Jun 1; 23 (11). ): 2968-80).

We tested whether the astrocyte-like cells of the present invention could be used in an in vitro disease model of Rett syndrome.
MECP2 protein in human adult fibroblast-derived astrocyte-like cells prepared by introducing NFIA, NICD and Tet2CD in Example 1 and lt-NES cell-derived astrocyte-like cells prepared in Example 4. In order to suppress the expression of shRNA, shRNA against the target sequence of MECP2 (SEQ ID NO: 24) was expressed by a lentivirus. In addition, a lentivirus expressing a mutant MECP2 that is not suppressed by the shRNA was introduced at the same time as the shRNA. These cells were cultured with E15.5 mouse-derived cerebral cortical neurons for 3 days. The results are shown in FIGS. 7 and 8.

Neurons seeded on astrocyte-like cells expressing shRNA for MECP2 significantly inhibited neurite outgrowth as compared to the control group. A neural tissue model in which astrocyte-like cells expressing shRNA for MECP2 and neurons are co-cultured is used as an in vitro disease model of Rett syndrome for elucidation of the molecular mechanism and pathogenesis of the disease and screening of therapeutic agents for the disease. Can be used for.

Example 9 Effect of p16INK4a on induction of differentiation of human adult fibroblasts into astrocyte-like cells A lentiviral vector incorporating the cDNA of p16INK4a (SEQ ID NO: 25) was prepared in the same manner as in Example 1 (1). .. In addition, an empty lentiviral vector in which the cDNA was not incorporated was prepared and used as a control.

The prepared lentiviral vector was prepared from the lentiviral vector incorporating the NFIA cDNA (SEQ ID NO: 1) prepared in Example 1 (1), the lentiviral vector incorporating the NICD cDNA (SEQ ID NO: 2), and Tet2CD. It was introduced into human adult skin fibroblasts (NHDF-Ad) (purchased from Lonza) together with cDNA (SEQ ID NO: 3) and cultured for 11 days. FIG. 9 shows the ratio of the number of GFAP-positive cells to the number of DAPI-positive cells after culturing, and the ratio of the number of GFAP-positive cells after culturing to the number of initial cells at the start of culturing. When p16INK4a was introduced, the efficiency of inducing differentiation of human adult fibroblasts into astrocyte-like cells was significantly reduced as compared with the control.

Example 10 Effect of knockdown of p16INK4a gene, p53 gene, p14ARF gene and p21 gene on induction of differentiation from human adult fibroblasts to astrosite-like cells Nucleotide sequence encoding shRNA on p14ARF gene (SEQ ID NOS: 14 and 15) Lentivirus vector incorporating the shRNA for the p53 gene, a lentivirus vector incorporating the shRNA encoding the p53 gene (SEQ ID NOs: 16 and 17), and a nucleic acid encoding the shRNA for the p21 gene (SEQ ID NOs: 18 and 19). It encodes a lentivirus vector, a lentivirus vector incorporating a nucleic acid encoding shRNA for the p16INK4a gene (SEQ ID NOs: 20 and 21), and a shRNA that targets a base sequence that is not present in the gene of human adult fibroblasts. A lentivirus vector (control) incorporating the nucleic acids (SEQ ID NOs: 22 and 23) was prepared in the same manner as in Example 1 (1).

The prepared lentiviral vector containing the NFIA cDNA (SEQ ID NO: 1) prepared in Example 1 (1), the lentiviral vector incorporating the NICD cDNA (SEQ ID NO: 2), and the lentiviral vector containing the NICD cDNA (SEQ ID NO: 2), respectively. It was introduced into human adult skin fibroblasts (NHDF-Ad) (purchased from Lonza) together with Tet2CD cDNA (SEQ ID NO: 3) and cultured for 11 days. The ratio of the number of GFAP-positive cells to the number of DAPI-positive cells after culturing, and the ratio of the number of GFAP-positive cells after culturing to the number of initial cells at the start of culturing are shown in FIGS. 10 to 13. Compared with the case of introducing a control shRNA, the efficiency of inducing differentiation from human adult fibroblasts to astrosite-like cells is higher when a shRNA that knocks down the p16INK4a gene, p53 gene, p14ARF gene and p21 gene is introduced. It increased significantly.

Reference Example 1 Mouse NFIA, NICD, Tet2CD, and REST / NRSF-VP16 were selected as candidates for inducing differentiation of mouse fibroblasts into astrocyte-like cells. A retrovirus vector containing nucleic acids encoding each of these inducing factors (SEQ ID NOS: 1-4, respectively) was prepared in the same manner as in Example 4. The retroviral vector was introduced into mouse embryonic fibroblasts (MEF). In the introduction, one of the above retrovirus vectors was used, or two or three of them were used in combination. The expression of GFAP was examined 4 days after the introduction of the factor. The proportion of GFAP-expressing cells is shown in FIG.

NFIA alone was sufficient as an inducing factor to induce differentiation of mouse fibroblasts into astrocyte-like cells. In addition, it was clarified that the differentiation induction efficiency was increased by combining NFIA with other factors.

The nucleic acid base sequences used in Examples and Reference Examples of the present application, as well as the amino acid sequences of peptides and the base sequences of nucleic acids that can be used in the present invention are shown below.

SEQ ID NO: 1: Nucleotide sequence of mouse NFIA cDNA. In the sequence, the underlined part indicates the HA tag sequence.

SEQ ID NO: 2: cDNA sequence of mouse NICD. In the sequence, the wavy part shows the Myc tag sequence and the underlined part shows the linker sequence.

SEQ ID NO: 3: Base sequence of cDNA of mouse Tet2CD. In the sequence, the underlined part indicates the FLAG tag sequence.

SEQ ID NO: 4: Base sequence of cDNA of mREST / NRSF-VP16. In the sequence, the broken line part shows the FLAG tag sequence, the underlined part shows the sequence encoding mREST / NRSF, the wavy line part shows the linker sequence, and the italic part shows the sequence encoding VP16.

SEQ ID NO: 5: Nucleotide sequence of human NFIA cDNA (NM_001145511.1).

SEQ ID NO: 6: Nucleotide sequence of human NICD cDNA (part of NM_017617.4).

SEQ ID NO: 7: Base sequence of cDNA of human Tet2CD (part of NM_001127208.2).

SEQ ID NO: 8: Amino acid sequence of mouse NFIA (NP_035035.1).

SEQ ID NO: 9: Amino acid sequence of mouse NICD (part of NP_032740.3).

SEQ ID NO: 10: Amino acid sequence of mouse Tet2CD (part of NP_001035490.2).

SEQ ID NO: 11: Amino acid sequence of human NFIA (NP_001138983.1).

SEQ ID NO: 12: Amino acid sequence of human NICD (NP_060087.3).

SEQ ID NO: 13: Amino acid sequence of human Tet2CD (NP_001120680.1).

SEQ ID NO: 14: Nucleotide sequence of the forward chain of DNA encoding shp14ARF. In the sequence, the uppercase part indicates the part complementary to the target.

SEQ ID NO: 15: Nucleotide sequence of the reverse strand of DNA encoding shp14ARF. In the sequence, the uppercase part indicates the part complementary to the target.

SEQ ID NO: 16: Nucleotide sequence of the forward chain of DNA encoding shp53. In the sequence, the uppercase part indicates the part complementary to the target.

SEQ ID NO: 17: Nucleotide sequence of the reverse strand of DNA encoding shp53. In the sequence, the uppercase part indicates the part complementary to the target.

SEQ ID NO: 18: Nucleotide sequence of the forward chain of DNA encoding shp21. In the sequence, the uppercase part indicates the part complementary to the target.

SEQ ID NO: 19: Nucleotide sequence of the reverse strand of DNA encoding shp21. In the sequence, the uppercase part indicates the part complementary to the target.

SEQ ID NO: 20: Nucleotide sequence of the forward chain of DNA encoding shp16INK4a. In the sequence, the uppercase part indicates the part complementary to the target.

SEQ ID NO: 21: Nucleotide sequence of the reverse strand of DNA encoding shp16INK4a. In the sequence, the uppercase part indicates the part complementary to the target.

SEQ ID NO: 22: Nucleotide sequence of the forward chain of the DNA encoding the shRNA used as a control in Example 10. In the sequence, the uppercase part indicates the part complementary to the target.

SEQ ID NO: 23: Nucleotide sequence of the reverse strand of the DNA encoding the shRNA used as a control in Example 10. In the sequence, the uppercase part indicates the part complementary to the target.

SEQ ID NO: 24: Nucleotide sequence in the MECP2 gene targeted by the shRNA used in Example 8.

SEQ ID NO: 25: Base sequence of cDNA of p16INK4a (NM_000077.4).

According to the method of the present invention, astrocyte-like cells having characteristics similar to those of astrocytes, which have been difficult to obtain in the past, can be obtained quickly, highly efficiently, and in large quantities from easily obtained human cells. In addition, an in vitro disease model can be constructed using the astrocyte-like cells and used for elucidation of the molecular mechanism and pathogenesis of the disease and screening of a therapeutic drug for the disease.

All publications and patent documents cited herein are incorporated herein by reference in their entirety. Although specific embodiments of the present specification have been described herein for purposes of illustration, those skilled in the art may make various modifications without departing from the spirit and scope of the present invention. Will be easily understood.

Claims (10)

A method for preparing astrocyte-like cells from human cells in vitro, in the following steps:
(1) A step of introducing at least one lentiviral vector containing a nucleic acid encoding at least two kinds of inducers into human cells; and (2) a step of inducing differentiation of the human cells to obtain astrocyte-like cells. Including
Here, the inducing factor comprises NFIA and comprises at least one of NICD and Tet2CD.
The human cells, a human fibroblast,
In the step (1), introduction of at least one lentiviral vector containing nucleic acids encoding NFIA, NICD and Tet2CD into human cells is excluded; and / or
In the step (1)
Whether to inhibit the expression of at least one gene selected from the p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene.
Inhibits at least one selected from p14ARF, p53, p21, p16INK4a and Rb.
Increase the expression of at least one gene selected from the MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, or activate at least one selected from MDM2, cyclin D1, CDK4, CDK6 and E2F. Including further to
The preparation method. The human cell is a cell derived from a patient with a nervous system disease, or a cell derived from a healthy person in which a mutation specifically present in the gene of the cell derived from the patient with a nervous system disease is artificially introduced into the gene. , The preparation method according to claim 1. An astrocyte-like cell prepared by the method according to any one of claims 1 and 2. A method for assessing the efficacy or toxicity of a test substance against nervous system diseases, the following steps:
(1) Claims 1 to 2 from cells derived from a patient with a nervous system disease or cells derived from a healthy person in which a mutation specifically present in the gene of a cell derived from the patient with a nervous system disease is artificially introduced into the gene. The step of preparing astrocyte-like cells by the method according to any one of the above; and (2) The effectiveness of the test substance by contacting the astrocyte-like cells obtained in step (1) with the test substance. Alternatively, the method comprising the step of evaluating toxicity. A method for assessing the efficacy or toxicity of a test substance against nervous system diseases, the following steps:
(1) A step of preparing astrocyte-like cells from cells derived from a healthy subject by the method according to any one of claims 1 and 2.
(2) In the astrocyte-like cells obtained in step (1), a step of knocking out or knocking down a gene related to a nervous system disease; and (3) the astrocyte-like cells obtained in step (2) and a test substance The method comprising contacting the test substance to evaluate the efficacy or toxicity of the test substance. An in vitro neural tissue model comprising the astrocyte-like cells and neurons of claim 3. A method for assessing the efficacy or toxicity of a test substance against nervous system diseases, the following steps:
(1) Claims 1 to 2 from cells derived from a patient with a nervous system disease or cells derived from a healthy person in which a mutation specifically present in the gene of the cell derived from the patient with a nervous system disease is artificially introduced into the gene. A step of preparing astrocyte-like cells by the method according to any one of the above;
(2) A step of co-culturing the astrocyte-like cells obtained in step (1) and neurons to prepare a nerve tissue model;
(3) The method according to the above method, which comprises a step of contacting the nerve tissue model obtained in step (2) with a test substance to evaluate the efficacy or toxicity of the test substance. A method for assessing the efficacy or toxicity of a test substance against nervous system diseases, the following steps:
(1) A step of preparing astrocyte-like cells from cells derived from a healthy subject by the method according to any one of claims 1 and 2.
(2) A step of knocking out or knocking down a gene related to a nervous system disease in the astrocyte-like cell obtained in the step (1);
(3) A step of co-culturing the astrocyte-like cells obtained in step (2) and neurons to prepare a nerve tissue model;
(4) The method according to the above method, which comprises a step of contacting the nerve tissue model obtained in step (3) with a test substance to evaluate the efficacy or toxicity of the test substance. An inducer from human cells to astrocyte-like cells comprising at least one lentiviral vector containing a nucleic acid encoding at least two inducers, wherein the inducer comprises NFIA. and wherein at least one of NICD and Tet2CD, the human cells, a human fibroblast,
Here, the inducer is excluded if it contains at least one lentiviral vector as a component, which comprises nucleic acids encoding NFIA, NICD and Tet2CD; and / or
An expression inhibitor of at least one gene selected from p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene, and / or at least one inhibitor selected from p14ARF, p53, p21, p16INK4a and Rb, and / Or an expression enhancer of at least one gene selected from MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, and / or at least one selected from MDM2, cyclin D1, CDK4, CDK6 and E2F. The inducer further comprising an activator. At least one lentiviral vector containing nucleic acids encoding at least two inducers; and
A kit for inducing human cells to astrocyte-like cells, which comprises an instruction manual describing the method according to any one of claims 1 and 2.
Here, the inducer comprises a NFIA, and comprises at least one of NICD and Tet2CD, the human cells, a human fibroblast,
Excluded here if the kit contains at least one lentiviral vector containing nucleic acids encoding NFIA, NICD and Tet2CD; and / or
An expression inhibitor of at least one gene selected from p14ARF gene, p53 gene, p21 gene, p16INK4a gene and Rb gene, and / or at least one inhibitor selected from p14ARF, p53, p21, p16INK4a and Rb, and / Or an expression enhancer of at least one gene selected from MDM2 gene, cyclin D1 gene, CDK4 gene, CDK6 gene and E2F gene, and / or at least one selected from MDM2, cyclin D1, CDK4, CDK6 and E2F. The kit further comprising an activator.

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