JPWO2019031595A1 - Method for selecting neural progenitor cells - Google Patents

Abstract

The present invention comprises (1) a step of introducing into a cell an mRNA in which a sequence encoding a marker protein is operably linked to a sequence recognized by miRNA expressed in neural progenitor cells, or a DNA encoding the mRNA. And (2) a method for selecting neural progenitor cells, comprising the step of selecting cells in which the expression of the marker protein is suppressed.

Description

The present invention relates to a method for selecting neural progenitor cells using mRNA having a sequence recognized by microRNA (hereinafter abbreviated as “miRNA”), and a method for producing neural progenitor cells using the selecting method.

Nerve cells are cells that perform information processing and information transmission in the nervous system, and have various shapes and types depending on their roles. Dopamine nerve cells, which are one type of nerve cells, are nerve cells that synthesize and release dopamine as a neurotransmitter, and are mainly present in the midbrain in the brain, and some of them are present in the hypothalamus. The dopamine neurons existing in the midbrain play an important role in the control of movements and emotions, and if these degenerate and drop out, serious neurodegenerative diseases such as Parkinson's disease can be caused. Cerebral cortex neurons, which are other nerve cells, are arranged in a regular layered structure in the cerebral cortex formed by nerve cell bodies, nerve fibers, glial cells, blood vessels and the like. It is known that the role of the cerebral cortex is highly differentiated depending on the site and extends to a wide variety of areas such as movement, somatosensory, vision, hearing and language. Degeneration and loss of cerebral cortical nerve cells can cause serious neurodegenerative diseases such as Alzheimer's disease. In order to treat neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, cell transplantation therapy for transplanting neural (progenitor) cells such as dopaminergic (progenitor) cells and cerebral cortical nerve (precursor) cells has been attracting attention. ..

As a method for obtaining such neural cells (including neural progenitor cells), for example, in the case of dopamine neural cells, stem cells such as induced pluripotent stem cells are transformed into floor plate cells that are dopaminergic neural progenitor cells. A method for inducing differentiation into dopaminergic neurons after differentiation is known (for example, Non-Patent Document 1). Further, in the case of cerebral cortical nerve cells, for example, a method of differentiating pluripotent stem cells into cerebral cortical nerve cells by a serum-free flocculation suspension culture (SFEBq) method is known (for example, Patent Document 1).

However, it has been reported that various problems occur when transplanting the nerve cells thus prepared. It has been reported that a tumor can be formed when a dopaminergic neural progenitor cell having a high proliferative capacity is transplanted (Non-Patent Document 2), and when a cell differentiated into the midbrain is clinically used, a serotonin-producing neuron It is also suggested that the graft may have caused a movement disorder due to the contamination of the bacterium (Non-patent Document 3). In order to suppress the side effects due to these transplants, it is important to increase the purity of desired cells in the donor cell population, and development of a method for purifying nerve cells is in progress. As such a method, for example, a method of selecting dopamine neural progenitor cells using Corin or NCAM/CD29, which is known as a cell surface antigen of floor plate cells and dopamine neurons, as an index has been reported (Non-Patent Documents 4 and 5). ). In the case of cerebral cortical neural progenitor cells, neuropilin-1 expression is used as an index in mouse fetal brain tissue to select cells characterized by axonal elongation, which is one of the characteristics of cerebral cortical neural progenitor cells. The method of doing is reported (nonpatent literature 6). However, in these methods, cell surface antigens are labeled with an antibody and sorted using a cell sorter, and a practical method for sorting neural progenitor cells is not known other than this method.

Here, the present inventors have previously been able to purify the cells by using miRNA specifically expressed in cell cultures, skeletal muscle progenitor cells, etc. as an index. It has been reported that it is not known and is useful as a method for purifying cells that cannot be sorted using an antibody (Patent Documents 2 and 3, Non-Patent Document 7).

JP 2016-5465 JP International Publication No. 2015/105172 International Publication No. 2016/114354

Kriks et al., Nature 480; 547-553 (2011) Doi D et al., Stem Cells 30(5); 935-945 (2012) Politis M et al., Sci Transl Med 2(38); 38ra46 (2010) Doi D et al., Stem Cell Reports 2; 337-350 (2014) Maria S et al., Stem Cells 31(8); 1548-1562 (2013) Sano N et al., Front Cell Neurosci 11; 123 (2017) Miki K et al., Cell Stem Cell 16(6): p699-711 (2015).

The present inventors have a problem that the method of selecting neural progenitor cells using an antibody against a surface antigen such as Corin and a cell sorter described above does not have sufficient cell selection efficiency and is difficult to scale up. I thought. Furthermore, since the above-mentioned method uses an antibody for cell selection, it is considered that there is a risk of contamination of residual antibody and a time-consuming and time-consuming problem when considering clinical use. .. Therefore, an object of the present invention is to provide a novel method for selecting neural progenitor cells that can solve the above problems, particularly a method that can be selected without using an antibody, and a method for producing neural progenitor cells using the method. ..

The present inventors attempted to develop a novel method for selecting neural progenitor cells in order to solve the above problems. The present inventors have identified a novel antigen by combining known cell surface antigens with respect to a selection method using a cell surface antigen and an antibody that recognizes it, which is a realistic method in the selection of neural progenitor cells. In this way, the idea was greatly changed from the general approach of improving the selection method, and it was suggested that neural progenitor cells could be selected by using a completely different approach in which the surface antigen is not used as an index. .. As one of such approaches, similar to the one used in the selection of skeletal muscle progenitor cells previously reported by the present inventors, miRNA was used as an index to intentionally select neural progenitor cells with known cell surface antigens. Thought to apply.

The present inventors first screened miRNAs expressed in neural progenitor cells based on the above idea. Then, focusing on miRNAs found by screening and miRNAs whose expression is known in neural progenitor cells, and including dopamine neural progenitor cells and cerebral cortical neural progenitor cells by using miRNA as an indicator rather than cell surface antigen Surprisingly, it is possible to select neural progenitor cells, and surprisingly, the cells selected in this way have the same surface as the conventional surface when the number of days from the initiation of pluripotent stem cell induction to the selection of neural progenitor cells is the same. The present inventors have found that the selection can be performed more efficiently than the method of selecting neural progenitor cells using an antigen as an index, and have completed the present invention.

That is, the present invention is as follows.
[1] A method for selecting neural progenitor cells, comprising steps (1) to (2) below:
(1) the sequence recognized by miRNA expressed in neural progenitor cells, mRNA functionally linked with a sequence encoding a marker protein, or the step of introducing a DNA encoding the mRNA into the cell, and
(2) A method comprising a step of selecting cells in which expression of the marker protein is suppressed.
[2] The method according to [1], wherein the neural progenitor cells are dopamine neural progenitor cells.
[3] The method according to [1], wherein the neural progenitor cells are cortical neural progenitor cells.
[4] The miRNA is miR-9-5p, miR-218-5p, miR-20a-5p, miR-124-3p, miR-7-5p, miR-135a-5p, let-7a-5p, let- 7g-5p, let-7i-5p, miR-26a-5p, miR-106a-5p, miR-320e, miR-374a-5p, let-7b-5p, let-7f-5p, miR-508-5p, miR-20b-5p, miR-30c-5p, miR-30d-5p, miR-30e-5p, miR-135b-3p, miR-17-5p, miR-330-3p, let-7d-5p, miR- 93-5p, miR-106b-5p, miR-335-5p, miR-98-5p, miR-92b-3p, miR-320c, miR-374b-5p, miR-320d, miR-4532, miR-584- 3p, miR-219-5p, miR-21-5p, miR-92a-3p, miR-449a, miR-18a-5p, miR-1908-3p, miR-210-5p, miR-34b-5p, miR- 34c-3p, miR-654-5p, miR-660-5p, miR-675-5p, miR-744-3p, miR-942-5p, miR-96-3p, miR-30a-5p, miR-491- The method according to [2], which is one or more miRNAs selected from the group consisting of 5p and miR-5001-5p.
[5] The miRNAs are miR-9-5p, let-7a-5p, let-7g-5p, let-7i-5p, miR-330-3p, let-7b-5p, let-7d-5p, let- 7f-5p, miR-135b-3p, miR-508-5p, miR-654-5p, miR-758-5p, miR-491-5p, miR-5001-5p, miR-191-5p, miR-20a- 5p, miR-342-3p, miR-99b-5p, miR-423-3p, miR-324-3p, miR-22-3p, miR-378a-3p, miR-296-5p, miR-99a-5p, miR-500a-3p, miR-345-5p, miR-335-5p, miR-134, miR-212-3p, miR-154-5p, miR-150-5p, miR-148a-3p, miR-382- 5p, miR-486-5p, miR-370, miR-373-3p, miR-512-5p, miR-516b-5p, miR-518b, miR-518c-5p, miR-519d, miR-523-3p, miR-526a, miR-98-5p, miR-140-3p, miR-378b, miR-128, miR-3180-3p, miR-1260a, miR-503-5p, miR-34c-5p, miR-3180, miR-21-3p, miR-4286, miR-4454, miR-138-5p, miR-1307-3p, miR-652-3p, miR-502-3p, miR-92b-5p, miR-501-3p, miR-1285-3p, miR-126-3p, miR-877-5p, miR-9-3p, miR-129-1-3p, miR-760, miR-365a-5p, miR-374a-5p, miR- 4532, miR-20b-3p, miR-363-5p, miR-339-3p, miR-513a-3p, miR-532-3p, miR-3180-5p, miR-4324, miR-1234-5p, miR- 329, miR-362-3p, miR-671-5p, miR-24-2-5p, miR-27a-5p, miR-411-5p, miR-197-5p, miR-222-5p, miR-584- 3p, miR-326, miR-133a, miR-17-5p, miR-92a-3p, miR-16-5p, miR-339-5p, mi R-331-3p, miR-203a, miR-298, miR-134-3p, miR-135a-3p, miR-138-1-3p, miR-144-5p, miR-154-3p, miR-15a- 5p, miR-15b-3p, miR-15b-5p, miR-16-1-3p, miR-181a-3p, miR-1910-3p, miR-1910-5p, miR-192-3p, miR-194- 3p, miR-194-5p, miR-196a-3p, miR-210-5p, miR-34c-3p, miR-450b-3p, miR-4536-3p, miR-4707-5p, miR-490-3p, miR-494-3p, miR-510-3p, miR-5196-3p, miR-542-5p, miR-574-3p, miR-589-3p, miR-642a-3p, miR-6503-5p, miR- One or more selected from the group consisting of 654-3p, miR-660-5p, miR-675-5p, miR-766-3p, miR-874-5p, miR-942-5p and miR-96-3p The method according to [3], which is miRNA.
[6] The miRNA group consisting of miR-9-5p, let-7a-5p, let-7g-5p, let-7i-5p, let-7b-5p, let-7f-5p and miR-508-5p The method according to any one of [1] to [5], which is one or more types of miRNA selected more.
[7] The method according to any one of [1] to [6], wherein the miRNA contains miR-9-5p.
[8] The mRNA comprises, in the 5′ to 3′ direction, one or more sequences recognized by the miRNA, a sequence encoding a marker protein, and one or more sequences recognized by the miRNA, The method according to any one of [1] to [7].
[9] The mRNA comprises, in the 5′ to 3′ direction, one sequence recognized by the miRNA, a sequence encoding a marker protein, and one or more sequences recognized by the miRNA. ].
[10] The mRNA comprises, in the 5′ to 3′ direction, one sequence recognized by miRNA, a sequence encoding a marker protein, and the same four sequences recognized by the miRNA. [9] The method described in.
[11] The method according to any one of [1] to [10], wherein the marker protein is a fluorescent protein or an apoptosis-inducing protein.
[12] The method according to [11], wherein the apoptosis-inducing protein is Bim.
[13] The method according to any one of [1] to [12], which further comprises a step of introducing a nucleic acid encoding a marker protein different from the marker protein into cells.
[14] The method according to [13], wherein the different marker proteins are fluorescent proteins or drug resistance proteins.
[15] The method according to any one of [1] to [14], wherein the neural progenitor cells are cells that have been induced to differentiate from pluripotent stem cells.
[16] The method according to [15], wherein the pluripotent stem cells are of human origin.
[17] The method according to [15] or [16], wherein the neural progenitor cells are selected 22 to 29 days after the initiation of differentiation induction.
[18] A method for producing neural progenitor cells from pluripotent stem cells, comprising the steps (1) to (3) below:
(1) a step of inducing differentiation of neural progenitor cells from pluripotent stem cells,
(2) mRNA obtained by functionally linking a sequence encoding a marker protein to a sequence recognized by miRNAs expressed in neural progenitor cells, or the mRNA obtained in the cell obtained in the step (1) The step of introducing DNA to
(3) A method for producing neural progenitor cells, which comprises a step of selecting cells in which the expression of the marker protein is suppressed.
[19] The method according to [18], which further comprises a step of introducing a nucleic acid encoding a marker protein different from the marker protein into cells.
[20] selected from the group consisting of miR-9-5p, let-7a-5p, let-7g-5p, let-7i-5p, let-7b-5p, let-7f-5p and miR-508-5p A kit for selecting neural progenitor cells, which comprises an mRNA containing a sequence recognized by a miRNA and a sequence encoding a marker protein, or a DNA encoding the mRNA.
[21] The mRNA comprises, in the 5′ to 3′ direction, one or more sequences recognized by the miRNA, a sequence encoding a marker protein, and one or more sequences recognized by the miRNA. The kit according to [20].
[22] The mRNA comprises, in the 5′ to 3′ direction, one sequence recognized by the miRNA, a sequence encoding a marker protein, and one or more sequence sequences recognized by the miRNA, 21].
[23] The mRNA contains, in the 5′ to 3′ direction, one sequence recognized by the miRNA, a sequence encoding a marker protein, and four identical sequences recognized by the miRNA. ] The kit described in.
[24] The kit according to any one of [20] to [23], further comprising a nucleic acid containing a sequence encoding a marker protein different from the marker protein.

According to the present invention, donor cells for cell transplantation used for treatment of Parkinson's disease, etc., which can be used as a tool for disease elucidation and/or a tool for drug development, can be used to select neural progenitor cells using miRNA. Become. In particular, the present invention is excellent in operability, and compared to the conventional method for selecting neural progenitor cells, the required labor, time and recovery efficiency can be significantly improved. Moreover, since the neural progenitor cells produced by the method of the present invention do not use an antibody for selection, there is no risk of residual antibody contamination and scale-up is easy.

FIG. 1a is a schematic diagram showing the flow of induction from iPS cells to midbrain dopamine neural progenitor cells and forebrain neurons. After performing the primary culture, the cells were subcultured on the 12th to 14th days, and the secondary culture was performed. Transfections were performed on day 21 or 28. FIG. 1b shows a flow of introducing a miRNA switch using neural progenitor cells differentiated from iPS cells and selecting by FACS or the like. This figure shows that the transfection is performed on the 21st day, but the transfection may be performed on the 14th or 28th day. Figure 2a shows the flow of 5'UTR, 3'UTR and ORF production. Figure 2b shows the flow of full length DNA fusion PCR. FIG. 2c shows the flow of template preparation of miR-responsive mRNA. FIG. 2d shows the flow of template preparation of tandem miRNA. FIG. 3 is a dot plot diagram when miRNA switch was introduced from iPS cells 14, 21, and 28 days after the initiation of differentiation induction, and FACS analysis was performed the next day. Using miRNA switches (miR-218-5p and miR-9-5p), the left figure on each day is the figure without the controls, and the right figure is the figure with the controls. The vertical axis represents BFP and the horizontal axis represents EGFP fluorescence amount. As a control, cells into which BFP and EGFP mRNA having no miRNA target sequence were introduced were used. FIG. 4 shows the expression levels of miRNA (miR-9-5p(a) and miR-218-5p(b)) in each of the cells (1231a3 and Kh1) induced to be differentiated into the midbrain and the forebrain as the induction of differentiation 0 It is the figure which investigated over time from the day to the 35th day. The numerical value on the vertical axis was standardized on the basis of the expression level of RNU6B. FIG. 5 shows that miR-218-5p switch (white bar) or miR-9-5p switch (grey bar) was introduced into dopaminergic neural progenitor cells that had been induced to differentiate from iPS cells for 21 days, and the cells were subjected to FACS selection the next day. It is the figure which investigated the expression change rate of a marker gene. The expression change rate represents the ratio of the average expression level of each gene in each miRNA-positive cell to each miRNA-negative cell as a common logarithm (n=4). Error bars indicate standard deviation. FIG. 6 is a diagram in which the expression change rate of each marker gene was examined in cells in which dopamine neural progenitor cells differentiated from iPS cells were subjected to FACS selection using the miR-9-5p switch. The miRNA switch was introduced on the 21st day (n=3) and 28th day (n=1) from the initiation of differentiation induction, and the FACS selection was performed the next day. The rate of expression change is expressed as the common logarithm of the ratio of the average expression level of each gene of miR-9-5p positive cells to miR-9-5p negative cells. Error bars indicate standard deviation. FIG. 7 is a diagram in which the expression change rate of each marker gene was investigated in the dopamine neural progenitor cells differentiated from iPS cells and subjected to FACS selection using the miR-218-5p switch. The miRNA switch was introduced on the 21st day (n=4) and 28th day (n=4) from the initiation of differentiation induction, and the FACS selection was performed the next day. The rate of change in expression is expressed as the ratio of the average expression level of each gene of miR-218-5p-positive cells to miR-218-5p-negative cells as a common logarithm. Error bars indicate standard deviation. FIG. 8 is a diagram in which the expression change rate of each marker gene in the cells obtained by FACS sorting the dopamine neural progenitor cells differentiated from iPS cells using an anti-CORIN antibody was examined. Analysis was performed on the 21st day (n=4) and 28th day (n=1) from the start of differentiation induction. The rate of change in expression is expressed as the common logarithm of the ratio of the average expression level of each gene in CORIN-positive cells to CORIN-negative cells. Error bars indicate standard deviation. Figure 9 shows that the miR-9-5p switch was introduced into dopaminergic neural progenitor cells that had been induced to differentiate from iPS cells for 21 days, and FACS sorting was performed the next day. It is the figure which carried out the immunostaining of the sample which carried out using the antibody which couple|bonds with each marker protein specifically. Comparisons were made with unsorted, miR-9-5p positive and miR-9-5p negative cells. FIG. 10 shows the results of comparative analysis of dopaminergic neural progenitor cells induced to differentiate from iPS cells using a tandem miR-9-5p switch and a normal miR-9-5p switch. The vertical axis represents the BFP fluorescence amount, and the horizontal axis represents the EGFP fluorescence amount. The upper dot plot shows a merged image of the control (Ctrl: red) and the reaction (blue) of each switch. As the Ctrl, cells into which BFP and EGFP mRNA having no miRNA target sequence were introduced were used. The lower row shows the contour plot of the analysis in which each switch was reacted. Figure 11 is a dot plot of the original miR-9-5p switch (left figure) and tandem miR-9-5p switch introduced into dopaminergic neural progenitor cells that have been induced to differentiate from iPS cells for 21 days, and then FACS sorted the next day. It is a figure. The vertical axis represents the BFP fluorescence amount, and the horizontal axis represents the EGFP fluorescence amount. FIG. 12a shows cells at 27 days and 20 days after initiation of differentiation induction of dopaminergic neural progenitor cells (1231A3 strain (human iPS cell line), Kh1 strain (human ES cell line)) differentiated from pluripotent stem cells. It is a figure which shows the viability of the cell in each concentration (0,1,5,10,15 microg/ml) of puromycin when it uses. As a comparative control, the cell viability when Triton is added at 1% is also shown. Blank shows fluorescence of only the plate, Control shows autofluorescence of cells not treated with CyQuant kit, and error bars show standard deviation. Figure 12b, when using the cells at day 27 and day 20 after the initiation of differentiation induction of dopamine neural progenitor cells (1231A3 strain, Kh1 strain) differentiated from iPS cells, the puromycin resistance gene was introduced (0, It is a figure which shows the survival rate of the cell at the time of (50, 75, 100 ng/ml). Puromycin was added to the medium at 10 μg/ml. As a comparative control, the survival rate of each cell without the addition of puromycin is shown. Blank indicates fluorescence only from the plate. Error bars indicate standard deviation. FIG. 12c is a diagram in which dopamine neural progenitor cells differentiated from iPS cells (1231A3 strain, Kh1 strain) were used for the cells 27 days and 20 days after the initiation of differentiation induction, and were not transfected, puromycin. It is a figure which shows the survival rate of the cell at the time of introduce|transducing a resistance gene (100 ng/ml) and further introducing Bim switch RNA (0, 50, 75, 100 ng/ml). Puromycin was added to the medium at 10 μg/ml. As a comparative control, the survival rate of each non-transfected cell in a puromycin-free medium (no selection) is shown. Error bars indicate standard deviation. Figure 13a (upper panel) shows the population that was not sorted with puromycin (left panel) and puromycin (right panel) that had Bim switch introduced into dopamine neural progenitor cells that had been induced to differentiate from iPS cells for 21 days. It is a photograph. The lower panel of FIG. 13a is a photograph of cell aggregates formed by further culturing the cells on a 96-well low-adhesion plate for 7 days. The scale bar is 100 μm in both figures. FIG. 13b shows the cell recovery efficiency when a BFP-miRNA switch or Bim switch was introduced into dopamine neural progenitor cells that had been induced to differentiate from iPS cells for 21 days, and FACS or Bim switch selection was performed, respectively. The vertical axis represents the ratio of the number of positive cells sorted by each method to the number of cells before transfection. Error bars indicate standard deviation. Figure 14 shows the introduction of Bim switch and puromycin-resistant mRNA into dopamine neural progenitor cells, which were induced to differentiate from iPS cells for 21 days, and cultured overnight in puromycin-supplemented medium. It is the figure which investigated the expression change rate of each marker gene in the cell clump which continued culture and was formed. The rate of change in expression is expressed as the common logarithm of the ratio of the average expression level of each gene in cells sorted by Bim switch to unsorted cells (n=3). Figure 15 shows that after the dopaminergic neural progenitor cells that had been induced to differentiate from iPS cells for 21 days were selected with a Bim switch, they were continuously cultured for 7 days on a 96-well low-adhesion plate to form cell clumps, and again with Accumax to form single cells. Dissociated, seeded on an 8-well chamber slide (iMatrix coat), fixed 7 days later (differentiation induction 35 days) with 4% PFA, and immunostained with an antibody that specifically binds to each marker protein. is there. Comparisons were made with unsorted cells and cells sorted with the Bim switch. FIG. 16a is a dot plot diagram when neural progenitor cells differentiated from mouse ES cells were introduced with the miR-9-5p switch 14 days after the initiation of differentiation induction and FACS sorted the next day. FIG. 16b is a diagram in which the expression rate of each cerebral cortical nerve cell marker gene in the cells subjected to FACS selection in FIG. 16a was examined. The vertical axis represents the rate of change in the expression level of each marker gene in miR-9-5p negative or miR-9-5p positive cells relative to unsorted cells. FIG. 16c is a diagram in which the expression rate of Notch signaling gene in the cells subjected to FACS selection in FIG. 16a was examined. The vertical axis represents the rate of change in the expression level of each marker gene in miR-9-5p negative or miR-9-5p positive cells relative to unsorted cells. FIG. 16d is a diagram in which the expression rates of the early mesoderm and neural crest marker genes in the cells subjected to FACS selection in FIG. 16a were examined. The vertical axis represents the rate of change in the expression level of each marker gene in miR-9-5p negative or miR-9-5p positive cells relative to unsorted cells.

1. Method for selecting neural progenitor cells The present invention includes (1) mRNA having responsiveness to miRNA expressed in neural progenitor cells (hereinafter sometimes abbreviated as "miRNA-responsive mRNA"), or encoding the mRNA A step of introducing DNA (hereinafter, these mRNA and DNA may be abbreviated as "miRNA-responsive nucleic acid" in a cell) into cells, and (2) using the suppression of expression of a marker protein encoded by the nucleic acid as an index The present invention provides a method for selecting neural progenitor cells (hereinafter, may be abbreviated as “selection method of the present invention”) including a step of selecting neural progenitor cells.

<Neural progenitor cells>
In the present specification, the “neural progenitor cell” means an undifferentiated cell having the ability to differentiate into a mature neural cell, and a cell population including the cell is also included in the “neural progenitor cell”. Examples of neural progenitor cells used in the present invention include dopamine neural progenitor cells, cerebral cortical neural progenitor cells, GABAergic neural progenitor cells, motor neural progenitor cells, and the like.

The animal from which the neural progenitor cells are derived is not particularly limited, and examples thereof include mammals (eg, mice, rats, hamsters, guinea pigs, dogs, monkeys, orangutans, chimpanzees, humans, etc.). In light, it is preferably human.

Examples of dopaminergic neural progenitor cells include, for example, bottom plate cells having the ability to differentiate into midbrain dopamine neurons, neuroectodermal cells characterized by expression markers such as intermediate filament protein Nestin, and the like, but preferably bottom plate cells. Is. As used herein, “floor plate cell” means a morphologically specialized organizer cell located in the ventral midline of the neural tube from the spinal cord to the diencephalon. Bottom plate cells are specific cell markers such as FOXA2 (forkhead box A2), LMX1A (LIM homeobox transcription factor 1 alpha), En1 (Engrailed homeobox 1), Nurr1 (Nuclear Receptor-related 1), OTX2 (orthodenticle homeobox 2) and Corin. Can be characterized by its expression, but does not necessarily mean that all of these markers are positive in vitro. Further, "dopaminergic nerve cells" means nerve cells that have the ability to produce dopamine (dopamine; 3,4-dihydroxyphenylethylamine), and dopaminergic nerve cells do not need to constantly produce dopamine, All you need is the ability. Mature midbrain dopamine neurons can be characterized in vitro by the expression of specific cell markers such as tyrosine hydroxylase (TH), FOXA2 and Nurr1, but not necessarily all of these markers are positive. do not do.

The cerebral cortex is located in the surface layers of the frontal lobe, parietal lobe, temporal lobe, and occipital lobe, and contains many nerve cells. The role of the cerebral cortex is highly differentiated depending on the site, and extends to a wide variety of areas such as movement, somatosensory, vision, hearing and language. As used herein, the term “cerebral cortical neural progenitor cell” refers to a cell capable of differentiating into any cortical neural cell having a wide variety of functions. Cerebral cortical neural progenitor cells can be characterized by the expression of specific cellular markers such as Pax6, Ctip2, Emx1 and Fezf, but in vitro do not necessarily mean that all of these markers are positive.

In the step of introducing the miRNA-responsive nucleic acid of the selection method of the present invention, the neural progenitor cell into which the miRNA-responsive nucleic acid is introduced and to be selected may be any cell population capable of containing neural progenitor cells. The origin of neural progenitor cells used in the present invention is not particularly limited, and may be neural progenitor cells that have been induced to differentiate from pluripotent stem cells, or may be cells taken out from the body, Cells that are differentiated from pluripotent stem cells are preferred. Therefore, the present invention also includes an embodiment in which the miRNA-responsive nucleic acid is introduced into a cell population in which it is unknown whether or not it contains neural progenitor cells. Further, the neural progenitor cells used in the present invention may be in the form of a cell population in which differentiated cells other than neural progenitor cells, progenitor cells of the cells, and the like are mixed, but preferably, only neural cell lineages It is a cell population consisting of cell types having the differentiating ability.

<miRNA-responsive mRNA>
As used herein, the term “miRNA-responsive mRNA” (sometimes referred to as “miRNA switch”) means a sequence recognized by a miRNA expressed (preferably specifically expressed) in neural progenitor cells. (Sometimes referred to as “miRNA target sequence”), and an mRNA that contains a sequence encoding a marker protein and is operably linked so that expression (translation) of the marker protein is controlled by the miRNA target sequence. means.

As used herein, being regulated by a miRNA target sequence correlates with the activation amount of the miRNA expressed in neural progenitor cells (that is, the amount of the miRNA that is mature and has the ability to recognize the target sequence). Thus, it means that the expression level of the marker protein is suppressed by miRNA-responsive mRNA being subjected to translational inhibition or degradation.

The “miRNA” in the present invention refers to a short-chain (usually 20-25 bases) non-coding RNA existing in a cell, which is involved in the regulation of gene expression through inhibition of translation of mRNA into protein and degradation of mRNA. means. This miRNA is transcribed from DNA as a single-stranded pri-miRNA capable of forming a hairpin loop structure containing the miRNA and its complementary strand, and a part of the pre-miRNA is cleaved by an enzyme called Drosha in the nucleus. After being transported to the outside of the nucleus, it is further cut by Dicer to act and function.

As used herein, the “miRNA expressed in neural progenitor cells” may be a miRNA that is highly expressed in neural progenitor cells to be sorted as compared with cells other than the neural progenitor cells. There is no particular limitation. For example, when selecting dopamine neural progenitor cells or cerebral cortical neural progenitor cells as neural progenitor cells, compared to cells other than dopamine neural progenitor cells or cerebral cortical neural progenitor cells, respectively, 10% or more, 20% or more, Examples include miRNAs that are highly expressed at a rate of 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or more. Such miRNAs can be found in miRNAs registered in database information (for example, http://www.mirbase.org/ or http://www.microrna.org/) and in literature information listed in the database. It may be appropriately selected from the described miRNAs, or may be newly identified using microarray analysis or the like.

The miRNA used as an index when selecting dopaminergic progenitor cells, miR-9-5p (in the case of human, represented by SEQ ID NO:731), miR-218-5p, miR-20a-5p, miR-124 -3p, miR-7-5p, miR-135a-5p, let-7a-5p, let-7g-5p, let-7i-5p, miR-26a-5p, miR-106a-5p, miR-320e, miR -374a-5p, let-7b-5p, let-7f-5p, miR-508-5p, miR-20b-5p, miR-30c-5p, miR-30d-5p, miR-30e-5p, miR-135b -3p, miR-17-5p, miR-330-3p, let-7d-5p, miR-93-5p, miR-106b-5p, miR-335-5p, miR-98-5p, miR-92b-3p , MiR-320c, miR-374b-5p, miR-320d, miR-4532, miR-584-3p, miR-219-5p, miR-21-5p, miR-92a-3p, miR-449a, miR-18a -5p, miR-1908-3p, miR-210-5p, miR-34b-5p, miR-34c-3p, miR-654-5p, miR-660-5p, miR-675-5p, miR-744-3p , MiR-942-5p, miR-96-3p, miR-30a-5p, miR-491-5p and miR-5001-5p are preferably one or more miRNAs selected from the group consisting of miR-9 -5p, miR-218-5p, miR-20a-5p, miR-124-3p, miR-7-5p, miR-135a-5p, let-7a-5p, let-7g-5p, let-7i-5p , MiR-26a-5p, miR-106a-5p, miR-320e, miR-374a-5p, let-7b-5p, let-7f-5p, and miR-508-5p. Is more preferred.

Examples of miRNA used as an index when selecting cerebral cortical neural progenitor cells include miR-9-5p, let-7a-5p, let-7g-5p, let-7i-5p, miR-330-3p, let-7b -5p, let-7d-5p, let-7f-5p, miR-135b-3p, miR-508-5p, miR-654-5p, miR-758-5p, miR-491-5p, miR-5001-5p , MiR-191-5p, miR-20a-5p, miR-342-3p, miR-99b-5p, miR-423-3p, miR-324-3p, miR-22-3p, miR-378a-3p, miR -296-5p, miR-99a-5p, miR-500a-3p, miR-345-5p, miR-335-5p, miR-134, miR-212-3p, miR-154-5p, miR-150-5p , MiR-148a-3p, miR-382-5p, miR-486-5p, miR-370, miR-373-3p, miR-512-5p, miR-516b-5p, miR-518b, miR-518c-5p , MiR-519d, miR-523-3p, miR-526a, miR-98-5p, miR-140-3p, miR-378b, miR-128, miR-3180-3p, miR-1260a, miR-503-5p , MiR-34c-5p, miR-3180, miR-21-3p, miR-4286, miR-4454, miR-138-5p, miR-1307-3p, miR-652-3p, miR-502-3p, miR -92b-5p, miR-501-3p, miR-1285-3p, miR-126-3p, miR-877-5p, miR-9-3p, miR-129-1-3p, miR-760, miR-365a -5p, miR-374a-5p, miR-4532, miR-20b-3p, miR-363-5p, miR-339-3p, miR-513a-3p, miR-532-3p, miR-3180-5p, miR -4324, miR-1234-5p, miR-329, miR-362-3p, miR-671-5p, miR-24-2-5p, miR-27a-5p, miR-411-5p, miR-197-5p , MiR-222-5p, miR-584-3p, miR-326, miR-133a, miR-17-5p, miR-92a-3p, m iR-16-5p, miR-339-5p, miR-331-3p, miR-203a, miR-298, miR-134-3p, miR-135a-3p, miR-138-1-3p, miR-144- 5p, miR-154-3p, miR-15a-5p, miR-15b-3p, miR-15b-5p, miR-16-1-3p, miR-181a-3p, miR-1910-3p, miR-1910- 5p, miR-192-3p, miR-194-3p, miR-194-5p, miR-196a-3p, miR-210-5p, miR-34c-3p, miR-450b-3p, miR-4536-3p, miR-4707-5p, miR-490-3p, miR-494-3p, miR-510-3p, miR-5196-3p, miR-542-5p, miR-574-3p, miR-589-3p, miR- 642a-3p, miR-6503-5p, miR-654-3p, miR-660-5p, miR-675-5p, miR-766-3p, miR-874-5p, miR-942-5p and miR-96- One or more miRNAs selected from the group consisting of 3p are preferable, and among them, miR-9-5p, let-7a-5p, let-7g-5p, let-7i-5p, miR-330-3p, let- 7b-5p, let-7d-5p, let-7f-5p, miR-135b-3p, miR-508-5p, miR-654-5p, miR-758-5p, miR-491-5p and miR-5001- More preferably, one or more miRNA selected from the group consisting of 5p.

Alternatively, as miRNA to be used as an index when selecting neural progenitor cells, miR-9-5p, let-7a-5p, let-7g-5p, let-7i-5p, let-7b-5p, let-7f- One or more miRNAs selected from the group consisting of 5p and miR-508-5p are also preferred, especially miR-9-5p.

In addition, for selecting neural progenitor cells, a miRNA-responsive mRNA containing a sequence recognized by one or more miRNAs selected from the group consisting of the miRNAs listed above, or a DNA encoding the mRNA (eg, bottom The above-mentioned vector) is provided. Among them, the miRNA is a group consisting of miR-9-5p, let-7a-5p, let-7g-5p, let-7i-5p, let-7b-5p, let-7f-5p and miR-508-5p. It is preferably selected more preferably. Specific miRNA-responsive mRNAs include, for example, a sequence represented by any of SEQ ID NOs: 1 to 642, a sequence encoding a marker protein (eg, tagBFP (SEQ ID NO: 747)), and a 3'UTR sequence. (Eg, the sequence represented by SEQ ID NO:732), and the like.

In the present specification, the expression of miRNA in neural progenitor cells and recognizing the target sequence means that in neural progenitor cells, the miRNA interacts with a plurality of predetermined proteins to form an RNA-induced silencing complex (RISC). The RISC binds to the target sequence.

In the present specification, the miRNA target sequence is preferably a sequence that is completely complementary to the miRNA, but the miRNA target sequence does not match the perfectly complementary sequence (mismatch) as long as it can be recognized by the miRNA. ) May be included. The mismatch from the sequence completely complementary to the miRNA may be a mismatch that can be normally recognized by the miRNA in a desired cell, and the original function in the cell in vivo is about 40 to 50% mismatch. Is also acceptable. Such a mismatch is not particularly limited, 1 base, 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, or 10 bases mismatch, or 1 of the entire recognition sequence. Examples include disparities of% or more, 5% or more, 10% or more, 20% or more, 30% or more, or 40%. Further, in particular, like the miRNA target sequence on the mRNA provided by the cell, in particular, to the portion other than the seed region, that is, corresponding to about 16 bases on the 3'side of the miRNA, the 5'side in the target sequence The region may include a number of mismatches, and the portion of the seed region may include no mismatches or 1-base, 2-base, or 3-base mismatches. Such a sequence may have a base length including the number of bases to which RISC specifically binds, the length is not particularly limited, and may be the same as the number of bases of miRNA, or more than the number of bases of miRNA. The number of bases may be large or small. The sequence is preferably 18 bases or more and less than 24 bases, more preferably 20 bases or more and less than 22 bases. In the present specification, the miRNA target sequence is a neural progenitor cell to be selected and a miRNA-responsive mRNA having the sequence is introduced into a cell other than the cell, and a marker protein corresponding only in the neural progenitor cell to be selected is selected. It may be appropriately determined and used by confirming that the expression is suppressed.

As used herein, the term “marker protein” means a protein that is translated in a cell and functions as a marker, which allows cell selection. Examples of such proteins include, but are not limited to, fluorescent proteins, luminescent proteins, proteins that assist fluorescence, luminescence or coloration, membrane proteins, apoptosis-inducing proteins, suicide proteins and the like.

In the present specification, the fluorescent proteins include blue fluorescent proteins such as Sirius, TagBFP, and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; TurboGFP, AcGFP, TagGFP, Azami-Green (for example, hmAG1), green fluorescent proteins such as ZsGreen, EmGFP, EGFP, GFP2, HyPer; yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana; KusabiraOrange (eg, hmKO2), Orange fluorescent proteins such as mOrange; TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, red fluorescent proteins such as mStrawberry; TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (for example, hdKeimaRed), mRas. , MPlum, and other near-infrared fluorescent proteins, but are not limited thereto.

In the present specification, examples of the photoprotein include aequorin, but the photoprotein is not limited thereto. Examples of proteins that assist fluorescence, luminescence or coloration include, but are not limited to, enzymes that decompose fluorescence, luminescence or coloration precursors such as luciferase, phosphatase, peroxidase and β-lactamase. Here, in the present specification, when a protein that assists fluorescence, luminescence or coloration is used as a marker, in the selection of neural progenitor cells, contacting a cell with a corresponding precursor or a precursor corresponding to an intracellular Can be done by introducing.

In the present specification, the apoptosis-inducing protein means a protein having an apoptosis-inducing activity on cells. For example, IκB, Smac/DIABLO, ICE, HtrA2/OMI, AIF, endonuclease G, Bax, Bak, Noxa, Hrk (harakiri), Mtd, Bim, Bad, Bid, PUMA, activated caspase-3, Fas, Tk, etc. Examples include, but are not limited to: Preferably, it is Bim (for example, represented by SEQ ID NO:751 (base sequence) and SEQ ID NO:752 (amino acid sequence)).

As used herein, a suicide protein means a protein whose expression in a cell is lethal to that cell. As used herein, a suicide protein may be one that causes cell death by itself (eg, diphtheria A toxin) or one that sensitizes cells to a particular drug (eg, Herpes simplex thymidine kinase sensitizes cells to antiviral compounds). Examples of suicide proteins include diphtheria A toxin, herpes simplex thymidine kinase (HSV-TK), carboxypeptidase G2 (CPG2), carboxylesterase (CA), cytosine deaminase (CD), cytochrome P450 (cyt-450), deoxycytidine kinase. (DCK), nitroreductase (NR), purine nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), varicella zoster virus thymidine kinase (VZV-TK), xanthine-guanine phosphoribosyl transferase (XGPRT), and the like. It is not limited to these.

As used herein, the marker protein may be provided with a localization signal. Examples of the localization signal include a nuclear localization signal, a cell membrane localization signal, a mitochondrial localization signal, a protein secretion signal, and the like. Specifically, the classical nuclear localization sequence (NLS), M9 Sequences, mitochondrial targeting sequences (MTS), endoplasmic reticulum translocation sequences can be mentioned, but not limited to. Such a localization signal is particularly advantageous when cells are selected on an image by imaging cytometry or the like described later.

In the present specification, the expression that a marker protein is operably linked so as to be controlled by the miRNA target sequence means that the open reading frame (including the initiation codon) encoding the marker protein is upstream. It means that at least one miRNA target sequence is provided in the located untranslated region (5′UTR), in the untranslated region located downstream (3′UTR), and/or in the open reading frame. The miRNA-responsive mRNA preferably has a cap structure (7-methylguanosine 5'phosphate), an open reading frame encoding a marker protein, and a poly A tail from the 5'end in the 5'to 3'direction. , Within the 5'UTR, within the 3'UTR, and/or in the open reading frame with at least one miRNA target sequence. The position of the miRNA target sequence in the mRNA may be 5'UTR, 3'UTR, or within the open reading frame (3' side of the start codon), and all of these may be targeted to the miRNA target. It may have an array. Thus, the number of miRNA target sequences may be 1, 2, 3, 4, 5, 6, 7, 7, 8 or more. The upper limit of the miRNA target sequence is also not limited as long as the miRNA-responsive mRNA functions, and the number of bases from the cap structure to the start codon, the number of bases from the stop codon to the start point of the poly A tail, etc. are taken into consideration. You can decide. In addition, multiple (eg, 2, 3, 4, 5, 6 or more) miRNA target sequences may be concatenated, and other sequences between miRNA target sequences may be used as long as miRNA can bind. May be included. Furthermore, the mRNA may have a structure in which one or more miRNA target sequences, a sequence encoding a marker protein, and one or more miRNA target sequences are arranged in this order. When using multiple miRNA target sequences, two or more types of sequences may be used, but it is preferable that all miRNA target sequences are the same sequence. In a preferred embodiment, the miRNA-responsive mRNA of the present invention has a structure containing one miRNA target sequence, a marker protein-encoding sequence and four identical miRNA target sequences.

The number of bases and the types of bases between the cap structure of the miRNA-responsive mRNA and the miRNA target sequence are not particularly limited as long as they do not form a stem structure or a three-dimensional structure. For example, the number of bases between the cap structure and the miRNA target sequence can be designed to be 0 to 50 bases, preferably 10 to 30 bases. The number of bases and the type of base between the miRNA target sequence and the start codon may be arbitrary as long as they do not form a stem structure or a three-dimensional structure, and the number of bases between the miRNA target sequence and the start codon is It can be designed with an arrangement of 0 to 50 bases, preferably 10 to 30 bases.

In the present specification, it is preferable that the start codon AUG does not exist in the miRNA target sequence in the miRNA-responsive mRNA. For example, if the miRNA target sequence is present in the 5'UTR and contains AUG in the target sequence, it will be in frame in relation to the sequence encoding the marker protein linked to the 3'side. It is preferable to be designed. Alternatively, when AUG is contained in the target sequence, AUG in the target sequence can be converted to GUG for use. Further, in order to minimize the influence of AUG in the target sequence, the location of the target sequence in the 5'UTR can be changed appropriately. For example, the number of bases between the cap structure and the AUG sequence in the target sequence is 0 to 60 bases, for example, 0 to 15 bases, 10 to 20 bases, 20 to 30 bases, 30 to 40 bases, 40 to 50 bases. , 50 to 60 bases can be designed.

In the present invention, the mRNA may contain modified bases such as pseudouridine and 5-methylcytidine in place of ordinary uridine and cytidine for the purpose of reducing cytotoxicity. The positions of the modified bases can be all or part independently of both uridine and cytidine, and when they are part, the positions can be random positions at an arbitrary ratio.

In the present specification, as the 5′UTR sequence in miRNA-responsive mRNA, for example,
5'-GGUUCCGCGAUCGCGGAUC CA GAUCCACCGGUCGCCACCAUG-3': SEQ ID NO: 735
5'-GGUUCCGCGAUCGCGGAUC CA GAUCACACCGGUCGCCACCAUG-3': SEQ ID NO:736
5'-GGUUCCGCGAUCGCGGAUC CA GAUCAACACCGGUCGCCACCAUG-3': SEQ ID NO:737
5'-GGUUCCGCGAUCGCGGAUC CA GAUCAAACACCGGUCGCCACCAUG-3': SEQ ID NO:738
5'-GGUUCCGCGAUCGCGGAUC CA GAUCACCGGUCGCCACCAUG-3': SEQ ID NO:739
5'-GGUUCCGCGAUCGCGGAUC CA GUAUCCACCGGUCGCCACCAUG-3': SEQ ID NO:740
5'-GGUUCCGCGAUCGCGGAUC CA GAUCCAAACACCGGUCGCCACCAUG-3': SEQ ID NO: 741,
5'-GGUUCCGCGAUCGCGGAUC CA GAAACACCGGUCGCCACCAUG-3': SEQ ID NO: 742
5'-GGUUCCGCGAUCGCGGAUC CA GAUACCGGUCGCCACCAUG-3': SEQ ID NO: 743,
5'-GGUUCCGCGAUCGCGGAUC CA GAAACACCGGUCGCCACCAUG-3': SEQ ID NO: 744
(AUG at the 3'end of each sequence represents a start codon) and the like, but are not limited to this sequence. The position of the miRNA target sequence in the 5′UTR sequence is also not limited, but it is preferable that the miRNA target sequence is present between the underlined C and A in the above sequence.

In the present specification, the downstream of the sequence encoding the marker protein in the miRNA-responsive mRNA (that is, 3′UTR) is particularly limited if the miRNA-responsive nucleic acid is mRNA and has a poly A sequence. but not, for example, 5'-UCUAGACCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGG-3 ': SEQ ID nO: 732 in the poly a sequence (e.g., 5'-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3': SEQ ID nO: 733), but can be exemplified sequences added, but is not limited to this arrangement. In addition, for example, the 3'UTR sequence contained in the tandem miR-9-5p switch is 5'-UCUAGACCUUCUGCGGGGC GACGAGCUGucauacagcuagauaaccaaagaucauacagcuagauaaccaaagaucauacagcuagauaaccaaagaucauacagcuagauaaccaaagaGCGGCCGCG UGAAUAAAGCCUG4AGAGGCCUGCGUGAAUAAAGCCUGAGUAGG. : SEQ ID NO: 734 includes those having a poly A sequence. Further, the position of the miRNA target sequence in the 3'UTR sequence is not limited. When the miRNA-responsive nucleic acid is DNA, it may have a sequence complementary to the poly A sequence, and may have a polyadenylation site in place of or together with the sequence.

The miRNA-responsive nucleic acid can be synthesized by those skilled in the art by any method known in genetic engineering, if the sequence is determined according to the above. In particular, it can be obtained by an in vitro synthesis method using a template DNA containing a promoter sequence as a template.

Before carrying out the screening method according to this embodiment, a screening step for examining the effectiveness of the screening may be carried out. Specifically, having 5'UTR as exemplified above, a plurality of candidate miRNA-responsive nucleic acids are prepared, and each is introduced into a neural progenitor cell population of known purity, and the effectiveness of selection High miRNA target sequences as well as miRNA-responsive nucleic acids can be determined.

<Step of introducing miRNA-responsive nucleic acid>
In the step of introducing the miRNA-responsive nucleic acid of the selection method of the present invention into cells, miRNA-responsive mRNA to be introduced into cells may be used alone, or two or more kinds, for example, three kinds, 4 In some cases, 5 types, 6 types, 7 types, or 8 or more types are used. For example, when two or more types of miRNA-responsive mRNAs are used, it is desirable that the miRNA-responsive mRNAs have different miRNA target sequences and marker proteins. When two or more miRNA-responsive mRNAs are used, the number of miRNA target sequences contained in the miRNA-responsive mRNA, the distance from the 5'end of the miRNA target sequence, and other structural features of the miRNA-responsive mRNA are , Each miRNA-responsive mRNA may be the same or different. Alternatively, it is possible to use miRNA-responsive mRNAs that have the same miRNA target sequence but different marker proteins. For example, it is also possible to use a nucleic acid containing a sequence encoding an apoptosis-inducing protein that signals through a different pathway, for example, a miRNA-responsive mRNA in which Fas and Bim are combined with the same miRNA target sequence. It can be expected that cells other than neural progenitor cells can be efficiently removed.

In the present specification, the step of introducing miRNA-responsive mRNA into cells includes, for example, lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, DEAE dextran method, microinjection method, gene gun method, reverse transfection. It is preferable to directly introduce one or more miRNA-responsive mRNAs into cells contained in the cells using a method or the like. When two or more different types of miRNA-responsive mRNAs are introduced, or as a control mRNA having a miRNA-responsive mRNA and a sequence encoding a marker protein different from the sequence encoding the marker protein in the mRNA described below ( Hereinafter, it is also referred to as a transfection control), it is preferable to co-introduce a plurality of mRNAs into cells. This is because the ratio of the two or more kinds of co-introduced mRNA in cells is maintained in each cell, and the activity ratio of the proteins expressed from these mRNAs is constant in the cell population. The introduction amount at this time varies depending on the cell population to be introduced, mRNA to be introduced, introduction method and kind of introduction reagent, and those skilled in the art can appropriately select these in order to obtain a desired expression amount.

Alternatively, the miRNA-responsive mRNA and/or transfection control (hereinafter abbreviated as “miRNA-responsive mRNA etc.”) is in the form of DNA encoding the miRNA-responsive mRNA etc. Transfecting mRNA and the like) may be introduced into the cell. In this case, for example, a virus, a plasmid, a vector such as an artificial chromosome, a lipofection method, a liposome method, a microinjection method, or the like can be introduced into cells. Examples of viral vectors include retrovirus vectors and lentivirus vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007. ), adenovirus vector (Science, 322, 945-949, 2008), adeno-associated virus vector, Sendai virus vector (WO 2010/008054) and the like. Examples of the artificial chromosome vector include human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC, PAC) and the like. As the plasmid, a plasmid for mammalian cells may be used (Science, 322:949-953, 2008). The vector can contain regulatory sequences such as a promoter, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site so that miRNA-responsive mRNA and the like can be expressed.

In the present specification, examples of the transfection control include mRNA containing a sequence encoding a marker protein other than the marker protein encoded by miRNA-responsive mRNA and having no miRNA target site. The marker protein expressed from the transfection control is preferably a protein that does not cause cell death due to the expression of the marker protein, and therefore, a fluorescent protein, a luminescent protein, a protein that assists fluorescence, luminescence or coloration, a membrane protein, drug resistance Proteins are preferably used.

As the fluorescent protein, luminescent protein, protein that assists fluorescence, luminescence or coloration expressed from the transfection control, those similar to the marker protein encoded by the miRNA-responsive mRNA described above can be used. Further, the “drug resistant protein” used in the present invention may be any protein as long as it imparts resistance to the corresponding drug. Examples include, but are not limited to, proteins encoded by antibiotic resistance genes. Examples of antibiotic resistance genes include kanamycin resistance gene, ampicillin resistance gene, puromycin (puromycin) resistance gene, blasticidin resistance gene, gentamicin resistance gene, kanamycin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene. Etc. Preferably, a puromycin resistance gene is used as the drug resistance gene. When puromycin is used as a drug, the concentration in the medium is preferably 1 μg/ml to 20 μg/ml, more preferably 5 μg/ml to 15 μg/ml, and further preferably 10 μg/ml.

The selection method according to one embodiment of the present invention more preferably includes the step of simultaneously introducing miRNA-responsive mRNA and a transfection control into target cells. Such a step can be preferably carried out by co-introduction of miRNA-responsive mRNA and a transfection control. By using a transfection control, it is possible to select cells in which the expression of a marker protein expressed from miRNA-responsive mRNA is suppressed as neural progenitor cells even when the efficiency of introduction of miRNA-responsive mRNA into cells is low. It will be possible. When using a transfection control, those skilled in the art can appropriately select the introduction amount of the transfection control in order to obtain a desired expression level. For example, when introducing an mRNA containing a sequence encoding a puromycin resistance protein (e.g., represented by SEQ ID NO: 753), 50 ng or more is preferable, 75 ng or more is more preferable, 100 ng or more per 1 ml of the culture solution. Example: 100 ng) is more preferred.

When a transfection control is used, the marker protein expressed from the transfection control is preferably different from the marker protein contained in miRNA-responsive mRNA. For example, when the marker protein contained in miRNA-responsive mRNA is an apoptosis-inducing protein, the marker protein contained in the transfection control can be a fluorescent protein. In this case, the expression of the apoptosis-inducing protein is suppressed by the action of miRNA and the cells survive and the cells labeled with the fluorescent protein can be selected as neural progenitor cells. Alternatively, the marker protein expressed from the transfection control may be a drug resistance protein. In this case, cells that survive by suppressing the expression of the apoptosis-inducing protein by the action of miRNA and that are present even in the medium containing the drug can be selected as neural progenitor cells.

On the other hand, when the marker protein contained in the miRNA-responsive mRNA and the marker protein contained in the transfection control are fluorescent proteins, it is preferable that the fluorescent wavelengths of both fluorescent proteins are different.

<Neural progenitor cell selection process>
In the selection method of the present invention, the step of selecting cells in which the expression of the marker protein encoded by miRNA-responsive mRNA is suppressed is carried out by detecting a signal from the marker protein using a predetermined detection device. be able to. Examples of the detection device include, but are not limited to, a flow cytometer, an imaging cytometer, a fluorescence microscope, a light emission microscope, and a CCD camera. As such a detection device, those suitable for those skilled in the art can be used depending on the marker protein. For example, when the marker is a fluorescent protein or a luminescent protein, it is possible to sort using a flow cytometer, and when the marker is a protein that assists fluorescence, luminescence or coloration, a microscope is used, Using a culture dish coated with a light-responsive cell culture device, cells that have been colored can be irradiated with light, and cells that have not been exposed can be separated from the culture dish, allowing selection to be performed. When the protein is a membrane-localized protein, a cell surface protein-specific detection reagent such as an antibody, and a marker protein quantification method using the above detection device are possible, and a magnetic cell separation device (MACS) It is possible to isolate cells that do not undergo the marker protein quantification process, and when the marker protein is a drug resistance gene, a method can be used to detect the expression of the marker protein by drug administration and isolate living cells. Is. When the marker protein is an apoptosis-inducing protein, miRNA-expressing cells decrease the expression level of the marker protein, so that cells expressing the miRNA selectively survive, and the single cell of the cell is not subjected to the marker protein quantification process. Can be separated.

The suppression of the expression of the marker protein means, for example, the expression level of the marker protein expressed in the cell population into which the control mRNA in which the miRNA recognition sequence has been removed (hereinafter referred to as “control mRNA”) has been introduced from the miRNA-responsive mRNA. In comparison, when the expression amount of the marker protein is decreased in the cell population into which the same amount of miRNA-responsive mRNA as the control mRNA is introduced, it can be evaluated that the expression of the marker protein is suppressed. Alternatively, when the marker protein is an apoptosis-inducing protein, when the survival rate of the cell population in which the miRNA-responsive mRNA in the same amount as the control mRNA is introduced is higher than that in the cell population in which the control mRNA is introduced, the marker It can be evaluated that the expression of the protein is suppressed. By using the transfection control, it is possible to accurately evaluate even a cell population in which miRNA-responsive mRNA was not introduced sufficiently uniformly. More specifically, it can be confirmed by the method used in the examples.

As used herein, selecting cells means isolating neural progenitor cells with higher purity than a cell population that has not undergone the step of selecting using miRNA-responsive mRNA. In the present invention, the purity of the selected cells is not particularly limited as long as it is highly pure as compared with the case where the step of selecting using miRNA-responsive mRNA is not performed, but is preferably 60% or more, 60 %, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Examples are 95%, 96%, 97%, 98%, 99% and 100%.

The timing of cell selection is not limited as long as neural progenitor cells can be selected.For example, when using dopamine neural progenitor cells that have been induced to differentiate from human pluripotent stem cells, the period from 7 days to 35 days after initiation of differentiation induction It is preferable to sort by, and more preferably between 15 and 29 days. As shown in Examples below, when carrying out the sorting method of the present invention, it is unexpected when the sorting is carried out between 22 days and 29 days instead of around 12 days which is generally performed in the prior art. It was also shown that the dopaminergic neural progenitor cells can be sorted more efficiently. Therefore, it is preferable to perform the selection within 22 to 29 days. Further, for example, when using the cerebral cortical neural progenitor cells differentiated from human pluripotent stem cells, it is preferable to select between 7 days and 70 days after the initiation of differentiation induction, and between 28 days and 49 days. It is more preferable to select.

The period from the introduction of miRNA-inducible mRNA into cells to the selection of cells is not particularly limited, but it is preferable to perform selection 4 hours to 3 days after the introduction, and more preferably 1 day after the introduction. preferable.

<Pluripotent stem cells>
The pluripotent stem cell used in the present invention is a stem cell having pluripotency capable of differentiating into all cells existing in the living body and also having proliferative ability. For example, but not limited to, embryonic stem (ES) cells, sperm stem (GS) cells, embryonic germ (EG) cells, induced pluripotent stem (iPS) cells, embryos derived from cloned embryos obtained by nuclear transfer Stem (ntES) cells, Muse cells, etc. are included. Preferred pluripotent stem cells are ES cells, iPS cells and ntES cells, and more preferably human ES cells and human iPS cells, more preferably human iPS cells, from the viewpoint of being used for treatment of neurodegenerative diseases and the like. Is a cell.

(A) Embryonic stem cell
An ES cell is a stem cell that is established from the inner cell mass of an early embryo (for example, a blastocyst) of a mammal such as a human or a mouse and has pluripotency and the ability to proliferate by self-renewal.

ES cells are embryo-derived stem cells that are derived from the inner cell mass of the blastocyst, which is the embryo after the morula, at the 8-cell stage of the fertilized egg. It has the ability and the ability to grow by self-replication. ES cells were found in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and ES cell lines were subsequently established in primates such as humans and monkeys (JA Thomson et al. al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod. ., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165).

The ES cells can be established by removing the inner cell mass from the blastocyst of the fertilized egg of the target animal and culturing the inner cell mass on a fibroblast feeder. Further, maintenance of cells by subculture, leukemia inhibitory factor (LIF), using a culture medium added with substances such as basic fibroblast growth factor (bFGF) It can be carried out. For the method of establishing and maintaining human and monkey ES cells, see, for example, USP 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. US A. 92:7844-7848; Thomson JA, et. al. (1998), Science. 282:1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222:273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I, et al. (2006), Nature. 444:481-485 and the like.

As a culture medium for producing ES cells, for example, DMEM/F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng/ml bFGF was used. Human ES cells can be maintained under a humid atmosphere of 37° C., 2% CO ₂ /98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26:215-224). Also, ES cells should be passaged every 3-4 days, where passage is for example 0.25% trypsin and 0.1 mg/ml collagenase IV in PBS containing 1 mM CaCl ₂ and 20% KSR. Can be done using.

ES cells can be generally selected by the Real-Time PCR method using the expression of gene markers such as alkaline phosphatase, Oct-3/4 and Nanog as an index. In particular, in the selection of human ES cells, OCT-3/4, NANOG, the expression of gene markers such as ECAD can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26:443. -452).

Human ES cell lines such as WA01 (H1) and WA09 (H9) are obtained from WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from Institute of Frontier Medical Sciences, Kyoto University (Kyoto, Japan). It is possible.

(B) Sperm Stem Cell Sperm stem cells are pluripotent stem cells derived from the testis, which are the origin of spermatogenesis. Similar to ES cells, this cell is capable of inducing differentiation into cells of various lineages, and has the property that, for example, when it is transplanted into a mouse blastocyst, a chimeric mouse can be produced (M. Kanatsu-Shinohara et al. 2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012). It is capable of self-renewal in culture medium containing glial cell line-derived neurotrophic factor (GDNF), and sperm can be obtained by repeated passage under the same culture conditions as ES cells. Stem cells can be obtained (Takebayashi, Masanori et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), 41-46, Yodosha (Tokyo, Japan)).

(C) Embryonic germ cells Embryonic germ cells are cells that are established from embryonic primordial germ cells and have pluripotency similar to ES cells, such as LIF, bFGF, stem cell factor, etc. Can be established by culturing primordial germ cells in the presence of the substance (Y. Matsui et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550. -551).

(D) induced pluripotent stem cells induced pluripotent stem cells (iPS cells) can be prepared by introducing a specific reprogramming factor into somatic cells in the form of DNA or protein, almost equivalent to ES cells , An artificial stem cell derived from somatic cells having pluripotency and proliferative ability by self-renewal (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26:101-106 (2008); International. Published WO 2007/069666). The reprogramming factor is a gene specifically expressed in ES cells, its gene product or non-coding RNA, or a gene that plays an important role in maintaining undifferentiation of ES cells, its gene product or non-coding RNA, or It may be composed of a low molecular weight compound. As a gene contained in the reprogramming factor, for example, Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15. -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, etc. are exemplified, and these reprogramming factors may be used alone or in combination. Examples of the combination of reprogramming factors include WO2007/069666, WO2008/118820, WO2009/007852, WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119, WO2009/079007, WO2009/091659, WO2009/101084, WO2009/. 101407, WO2009/102983, WO2009/114949, WO2009/117439, WO2009/126250, WO2009/126251, WO2009/126655, WO2009/157593, WO2010/009015, WO2010/033906, WO2010/033920, WO2010/042800, WO2010/050626, WO2010/056831, WO2010/068955, WO2010/098419, WO2010/102267, WO2010/111409, WO 2010/111422, WO2010/115050, WO2010/124290, WO2010/147395, WO2010/147612, Huangfu D, et al. (2008) , Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474. , Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, RL Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106:8912-8917. , Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell. Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720. It is illustrated.

The reprogramming factors include histone deacetylase (HDAC) inhibitors [eg, valproic acid (VPA), trichostatin A, sodium butyrate, small molecule inhibitors such as MC 1293, M344, siRNA and shRNA against HDAC (eg, , HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene) etc.), MEK inhibitors (eg PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen synthase kinase- 3 inhibitors (eg Bio and CHIR99021), DNA methyltransferase inhibitors (eg 5-azacytidine), histone methyltransferase inhibitors (eg small molecule inhibitors such as BIX-01294, Suv39hl, Suv39h2, SetDBl and G9a Nucleic acid expression inhibitors such as siRNA and shRNA), L-channel calcium agonist (for example, Bayk8644), butyric acid, TGF-β inhibitor or ALK5 inhibitor (for example, LY364947, SB431542, 616453 and A-83-01), p53 inhibitors (eg siRNA and shRNA against p53), ARID3A inhibitors (eg siRNA and shRNA against ARID3A), miR-291-3p, miR-294, miR-295 and mir-302 miRNA, Wnt Signaling (eg soluble Wnt3a), neuropeptide Y, prostaglandins (eg, prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2, DMRTBl etc. The factors used for the purpose of improving the establishment efficiency are not particularly distinguished from the reprogramming factors in the present specification.

When in the form of a protein, the reprogramming factor may be introduced into the somatic cell by a technique such as lipofection, fusion with a cell membrane-penetrating peptide (eg, HIV-derived TAT and polyarginine), and microinjection.
On the other hand, in the case of DNA, for example, the same vector as in the method for introducing miRNA-responsive mRNA or the like can be used. The vector can contain a regulatory sequence such as a promoter, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site so that the nuclear reprogramming substance can be expressed, and further, if necessary, a drug resistance gene ( For example, selectable marker sequences such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene), thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG, etc. Can be included. In addition, the vector has LoxP sequences before and after the gene or promoter coding for the reprogramming factor and the gene coding for the reprogramming factor binding to it, after excision into the somatic cell. May be.

In the case of RNA, it can be introduced in the same manner as miRNA-responsive mRNA and the like.

As a culture medium for iPS cell induction, for example, DMEM containing 10 to 15% FBS, DMEM/F12 or DME culture medium (these culture medium further includes LIF, penicillin/streptomycin, puromycin, L- Glutamine, non-essential amino acids, β-mercaptoethanol and the like can be appropriately contained.) or commercially available culture medium [eg, mouse ES cell culture medium (TX-WES culture medium, Thrombo X), primate ES) Cell culture medium (primate ES/iPS cell culture medium, Reprocell), serum-free medium (mTeSR, Stemcell Technology)] and the like are included.

Examples of the culturing method include, for example, in the presence of 5% CO ₂ at 37° C., somatic cells and reprogramming factors are contacted on DMEM or DMEM/F12 culture medium containing 10% FBS, and the cells are cultured for about 4 to 7 days. Then, the cells are re-seeded on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.), and about 10 days after contact of somatic cells and reprogramming factors, with bFGF-containing primate ES cell culture medium. It can be cultured and allowed to develop iPS-like colonies about 30 to about 45 days or more after the contact.

Alternatively, at 37° C., in the presence of 5% CO ₂ , 10% FBS-containing DMEM culture medium on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.) (this further includes LIF, penicillin/streptomycin, It may contain puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc. as appropriate) to produce ES-like colonies after about 25 to about 30 days or more. .. Desirably, somatic cells to be reprogrammed are used instead of feeder cells (Takahashi K, et al. (2009), PLoS One. 4:e8067 or WO2010/137746), or extracellular matrix (for example, Laminin- 5 (WO2009/123349) and Matrigel (BD) are exemplified.
In addition to this, a method of culturing using a medium containing no serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci US A. 106:15720-15725). Furthermore, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) in order to improve the efficiency of establishment (Yoshida Y, et al. (2009), Cell Stem Cell. 5:237. -241 or WO2010/013845).

During the above culture, the culture medium is exchanged with a fresh culture medium once a day from the second day after the start of the culture. The number of somatic cells used for nuclear reprogramming is not limited, but is in the range of about 5×10 ³ to about 5×10 ⁶ cells per 100 cm ^{2 of} the culture dish.

The iPS cells can be selected according to the shape of the formed colonies. On the other hand, when a drug resistance gene that is expressed in association with a gene that is expressed when somatic cells are reprogrammed (eg, Oct3/4, Nanog) is introduced as a marker gene, a culture solution containing the corresponding drug (selection The established iPS cells can be selected by culturing with a culture medium). When the marker gene is a fluorescent protein gene, iPS cells are selected by observing with a fluorescence microscope, in the case of a luminescent enzyme gene, by adding a luminescent substrate, and in the case of a chromogenic enzyme gene, by adding a chromogenic substrate. can do.

As used herein, the term “somatic cell” refers to any animal cell (preferably mammalian cell including human) except germ line cells such as ova, oocytes, ES cells or totipotent cells. Somatic cells include, but are not limited to, both fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature, healthy or diseased somatic cells, and also primary culture cells. , Passage cells, and established cells are also included. Specifically, somatic cells include, for example, (1) neural stem cells, hematopoietic stem cells, mesenchymal stem cells, tissue stem cells (somatic stem cells) such as dental pulp stem cells, (2) tissue progenitor cells, (3) lymphocytes, epithelium Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells And differentiated cells such as adipocytes.

In the present invention, the mammalian individual from which somatic cells are collected is not particularly limited, but is preferably human. When the obtained iPS cells are used for regenerative medicine in humans, somatic cells should be collected from the patient himself or another person with the same or substantially the same HLA type from the viewpoint that no rejection reaction occurs. Is particularly preferable. Here, the type of HLA is "substantially the same", by using an immunosuppressant or the like, when the cells obtained by inducing differentiation from the somatic cell-derived iPS cells are transplanted into a patient, the transplanted cells are It means that the HLA types are matched to the extent that they can be engrafted. For example, the case where the main HLA (for example, 3 loci of HLA-A, HLA-B and HLA-DR, or 4 loci including HLA-C) is the same (hereinafter the same) can be mentioned.

(E) Clonal embryo-derived ES cells obtained by nuclear transfer
The ntES cell is a cloned embryo-derived ES cell produced by a nuclear transfer technique, and has almost the same characteristics as a fertilized egg-derived ES cell (T. Wakayama et al. (2001), Science, 292:. 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; J. Byrne et al. (2007), Nature, 450:497-502). That is, an ES cell established from an inner cell mass of a blastocyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell is an ntES (nuclear transfer ES) cell. For the production of ntES cells, a combination of a nuclear transfer technique (JB Cibelli et al. (1998), Nature Biotechnol., 16:642-646) and an ES cell production technique (above) is used (Seika Wakayama. Et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pages 47-52). In nuclear transfer, somatic cell nuclei are injected into unfertilized ova of a mammal that have undergone enucleation, and can be initialized by culturing for several hours.

(F) Multilineage-differentiating Stress Enduring cells (Muse cells)
Muse cells are pluripotent stem cells produced by the method described in WO2011/007900, specifically, trypsin treatment of fibroblasts or bone marrow stromal cells for a long time, preferably 8 hours or 16 hours trypsin. It is a pluripotent cell obtained by suspension culture after treatment and is positive for SSEA-3 and CD105.

2. Method for producing neural progenitor cells The present invention also includes a step of inducing differentiation of neural progenitor cells from pluripotent stem cells before step (1) of the selection method of the present invention, a method for producing neural progenitor cells (hereinafter referred to as " The production method of the present invention" may be abbreviated).

2-1. Method for inducing differentiation of dopamine neural progenitor cells The method for obtaining the dopamine neural progenitor cells used in the present invention is not particularly limited and may be directly collected from the living body, but from the viewpoint of obtaining a large amount of dopamine neural progenitor cells. It is preferable to induce differentiation using pluripotent stem cells as a starting material.
The method for inducing differentiation of dopaminergic neural progenitor cells from pluripotent stem cells is not particularly limited, but, for example, (i) pluripotent stem cells can be selected from BMP inhibitor, TGFβ inhibitor, SHH signal stimulant, FGF8 and GSK3β inhibitor. Adherent culture on an extracellular matrix in a culture medium containing a reagent selected from the group consisting of: (ii) suspension culture of the cells obtained in the step (i) in a culture medium containing a neurotrophic factor A method of inducing differentiation of dopaminergic neural progenitor cells according to the step (WO2015/034012).

<Extracellular matrix>
In the present specification, the “extracellular matrix” means a supramolecular structure existing outside the cell, which may be derived from nature or an artificial product (recombinant). Examples thereof include substances such as collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin and laminin, or fragments thereof. These extracellular matrices may be used in combination, eg preparations from cells such as BD Matrigel™. Laminin or a fragment thereof is preferable. In the present invention, laminin is a protein having a heterotrimeric structure having one α chain, one β chain, and one γ chain, and is not particularly limited. For example, α chain is α1, α2, α3, α4. Or α5, the β chain is β1, β2 or β3, and the γ chain is γ1, γ2 or γ3. More preferably, it is laminin 511 consisting of α5, β1 and γ1. In the present invention, laminin may be a fragment and is not particularly limited as long as it is a fragment having integrin-binding activity, for example, E8 fragment which is a fragment obtained by digestion with elastase. Good. Therefore, in the present invention, laminin 511E8 (preferably human laminin 511E8) described in WO2011/043405 is exemplified.

<BMP inhibitor>
Examples of the BMP inhibitor used in the present invention include proteinaceous inhibitors such as Chordin, Noggin, Follistatin, and Dorsomorphin (that is, 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3- pyridin-4-yl-pyrazolo[1,5-a]pyrimidine) and its derivatives (PB Yu et al. (2007), Circulation, 116:II_60; PB Yu et al. (2008), Nat. Chem. Biol. , 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904) and LDN193189 (ie 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo [1,5-a]pyrimidin-3-yl)quinoline) and the like. Dorsomorphin and LDN193189 are commercially available and are available from Sigma-Aldrich and Stemgent, respectively. LDN193189 is preferable.

The concentration of LDN193189 in the culture medium is not particularly limited as long as it is a concentration that inhibits BMP, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, It is, but not limited to, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM. Preferably, it is 100 nM.

<TGFβ inhibitor>
In the present specification, the “TGFβ inhibitor” means a substance that inhibits signal transduction from binding of TGFβ to a receptor and subsequent to SMAD, a substance that inhibits binding to a receptor ALK family, or Examples include substances that inhibit phosphorylation of SMAD by the ALK family, for example, Lefty-1 (NCBI Accession No. is exemplified by mouse: NM_010094, human: NM_020997), SB431542, SB202190 (above, RKLindemann et al. , Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (WO 2009146408) and derivatives thereof. Are exemplified. A83-01 is preferred.

The concentration of A83-01 in the culture medium is not particularly limited as long as it is a concentration that inhibits ALK5, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM, but not limited thereto. It is preferably 500 nM to 5 μM, more preferably 500 nM.

<SHH signal stimulant>
As used herein, the term “SHH (Sonic hedgehog) signal stimulant” causes deinhibition of Smoothened (Smo) caused by binding of SHH to a receptor Patched (Ptch1) and further activation of Gli2. Means a substance, for example, SHH, Hh-Ag1.5 (Li, X., et al., Nature Biotechnology, 23, 215-221 (2005).), Smoothened Agonist, SAG (N-Methyl-N'- (3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane), 20a-hydroxycholesterol, Purmorphamine and their derivatives are exemplified (Stanton BZ, Peng LF ., Mol Biosyst. 6:44-54, 2010). Preferred is Purmorphamine.

The concentration of Purmorphamine in the culture solution is not particularly limited as long as it is a concentration that activates Gli2, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM. , 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM, but not limited thereto. It is preferably 2 μM.

<GSK3β inhibitor>
In the present specification, the “GSK3β inhibitor” is defined as a substance that inhibits the kinase activity of GSK-3β protein (eg, phosphorylation ability for β-catenin), and many are already known. , Indirubin derivative BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin 3'-oxime), maleimide derivative SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl- 1H-indol-3-yl)-1H-pyrrole-2,5-dione), GSK-3β inhibitor VII (4-dibromoacetophenone), which is a phenyl α-bromomethylketone compound, and a cell membrane-permeable phosphorylated peptide L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQpSP-NH ₂ (SEQ ID NO: 1)) and CHIR99021 (6-[2-[4-(2,4-Dichlorophenyl)) with high selectivity -5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile). These compounds are commercially available, for example, from Calbiochem, Biomol, etc. and can be easily used, but they may be obtained from other sources or may be prepared by themselves. The GSK-3β inhibitor used in the present invention is preferably CHIR99021.

The concentration of CHIR99021 in the culture medium is, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM. , 40 μM, 50 μM, but not limited thereto. Preferably, it is 1 μM.

<FGF8>
The FGF8 used in the present invention is not particularly limited, but in the case of human FGF8, four splicing forms of FGF8a, FGF8b, FGF8e or FGF8f can be mentioned, and FGF8b is preferable. FGF8 is commercially available from, for example, Wako or R&D systems, and can be easily used, but it can also be obtained by forcedly expressing it in cells by a method known to those skilled in the art.

The concentration of FGF8 in the culture medium is, for example, 1 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 150 ng/ml, 200 ng/ml, 250 ng/ml, 500 ng/ml, 1000 ng/ml. ml, 2000 ng/ml, 5000 ng/ml, but not limited to these. It is preferably 100 ng/ml.

<Neurotrophic factor>
As used herein, the term “neurotrophic factor” means a ligand for a membrane receptor that plays an important role in survival and function maintenance of motor neurons, and includes, for example, Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin 3 (NT-3), Neurotrophin 4/5 (NT-4/5), Neurotrophin 6 (NT-6), basic FGF, acidic FGF, FGF-5, Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Insulin, Insulin Like Growth Factor 1 (IGF 1), Insulin Like Growth Factor 2 (IGF 2), Glia cell line-derived Neurotrophic Factor (GDNF), TGF-b2, TGF-b3, Interleukin 6 (IL-6), Ciliary Neurotrophic Factor (CNTF) and LIF. The preferred neurotrophic factor is GDNF and/or BDNF. The neurotrophic factor is commercially available from, for example, Wako or R&D systems, and can be easily used, but it can also be obtained by forcedly expressing it in cells by a method known to those skilled in the art.

The concentration of GDNF1 in the culture solution is, for example, 0.1 ng/ml, 0.5 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml. , 40 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml, 500 ng/ml, but are not limited thereto. It is preferably 10 ng/ml.

The concentration of BDNF1 in the culture solution is, for example, 0.1 ng/ml, 0.5 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml. , 40 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml, 500 ng/ml, but are not limited thereto. It is preferably 20 ng/ml.

<Process (i)>
The culture medium used in step (i) can be prepared using a medium used for culturing animal cells as a basal medium. As the basal medium, for example, Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640. The medium, Fischer's medium, Neurobasal Medium (Life Technologies), a mixed medium thereof and the like are included. Preferred is GMEM medium. The medium may contain serum or may be serum-free. If necessary, the medium includes, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute of FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen. It may contain one or more serum substitutes such as precursors, trace elements, 2-mercaptoethanol, 3'-thiol glycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts and the like. A preferred culture medium is a GMEM medium containing KSR, 2-mercaptoethanol, non-essential amino acids and pyruvic acid. It is possible to culture by appropriately adding a reagent selected from the group consisting of BMP inhibitor, TGFβ inhibitor, SHH signal stimulant, FGF8 and GSK3β inhibitor to this culture solution.

In the step (i), the adherent culture on the extracellular matrix can be carried out by culturing using an incubation vessel coated with the extracellular matrix. The coating treatment can be performed by placing a solution containing an extracellular matrix in a culture vessel and then removing the solution as appropriate.

The culture period of step (i) is not particularly limited, but it is desirable that the culture is performed for at least 10 days. More preferably, it is 12 to 21 days, still more preferably 12 to 14 days.

Regarding the culture conditions of step (i), the culture temperature is not particularly limited, but is about 30 to 40° C., preferably about 37° C., the culture is performed under an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is It is preferably about 2-5%. O ₂ concentration may be O ₂ concentration in normal air, or generally may be less hypoxic conditions even at high oxygen conditions than usual. In the present specification, the high oxygen condition is exemplified by an O ₂ concentration of 25% or more, an O ₂ concentration of 30% or more, an O ₂ concentration of 35% or more, or an O ₂ concentration of 40% or more. The hypoxic conditions, 10% or less of O ₂ concentration, O ₂ concentration of 5% or less, 4% or less of O ₂ concentration, O ₂ concentration of 3%, 2% or less O ₂ concentration, or 1% or less The O ₂ concentration of is exemplified.

<Step (ii)>
The culture medium used in step (ii) can be prepared by using a medium used for culturing animal cells as a basal medium. Examples of the basal medium include the same as those described in step (i) above. Preferably, it is Neurobasal Medium. The medium may contain serum or may be serum-free. If necessary, the medium includes, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute of FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen. It may contain one or more serum substitutes such as precursors, trace elements, 2-mercaptoethanol, 3'-thiol glycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, nucleic acids such as Dibutyryl cyclic AMP (dbcAMP). A preferred culture medium is Neurobasal Medium containing B27 supplement, ascorbic acid and dbcAMP. Neurotrophic factors can be appropriately added to this culture medium for culturing.

In the step (ii), the floating culture is culturing cells in a non-adhesive state in a culture vessel, and is not particularly limited, but is artificially treated for the purpose of improving the adhesiveness with cells (for example, A culture container that is not coated with an extracellular matrix or the like, or a treatment that artificially suppresses adhesion (for example, polyhydroxyethylmethacrylic acid (poly-HEMA), nonionic surface-active polyol (Pluronic F-127). Etc.) or a phospholipid-like structure (for example, a water-soluble polymer (Lipidure) having 2-methacryloyloxyethylphosphorylcholine as a constitutional unit) is used for the coating treatment.

The culture period of step (ii) is not particularly limited, but it is desirable that the culture period be at least 7 days. It is more preferably 7 days to 30 days, still more preferably 14 days to 21 days, or 14 days to 16 days, and even more preferably 16 days.

The culture conditions (culture temperature, CO ₂ concentration, O ₂ concentration) in step (ii) are the same as the culture conditions in step (i).

2-2. Method for inducing differentiation of cerebral cortical neural progenitor cells The method for obtaining cerebral cortical neural progenitor cells used in the present invention is not particularly limited and may be directly collected from a living body, but cerebral cortical neural progenitor cells are obtained in large quantities From this point of view, it is preferable to produce pluripotent stem cells as a starting material.
The method for inducing differentiation of cerebral cortical neural progenitor cells from pluripotent stem cells is not particularly limited, but, for example, (i) culture of pluripotent stem cells containing TGFβ inhibitor, bFGF, Wnt inhibitor and/or BMP inhibitor Examples thereof include a method of inducing differentiation of cerebral cortical neural progenitor cells by a step of suspension culture in a liquid, and (ii) a step of further culturing the cells obtained in the step (i) (WO2016/167372).

<TGFβ inhibitor>
The “TGFβ inhibitor” is as described above, and examples of the TGFβ inhibitor include the same ones as those described in the method for producing dopamine neural progenitor cells. The TGFβ inhibitor used in this step is preferably SB431542 or A-83-01.

The concentration of SB431542 in the culture medium is not particularly limited as long as it is a concentration that inhibits ALK5, for example, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 500 μM, 1 mM, but not limited to these. It is preferably 1 μM to 100 μM, more preferably 10 μM. The concentration of A-83-01 in the culture medium is not particularly limited as long as it is a concentration that inhibits ALK5, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM, but not limited thereto. It is preferably 500 nM to 5 μM, more preferably 500 nM to 2 μM.

<bFGF>
In the present specification, bFGF is also referred to as FGF2, and it is commercially available from, for example, Wako or Invitrogen and can be easily used, but it can also be obtained by forced expression into cells by a method known to those skilled in the art. it can.

The concentration of bFGF in the culture medium is, for example, 0.1 ng/ml, 0.5 ng/ml, 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng. /Ml, 9ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, 500ng/ml , 1000 ng/ml, but not limited to these. It is preferably 1 ng/ml to 100 ng/ml, more preferably 10 ng/ml.

<Wnt inhibitor>
As used herein, the term "Wnt inhibitor" refers to a substance that suppresses the production of Wnt, or a substance that inhibits the signal transduction that follows the binding of Wnt to the receptor and the accumulation of β-catenin. Examples include a substance that inhibits binding to a certain Frizzled family, a substance that promotes the degradation of β-catenin, and the like. Such Wnt inhibitors include, for example, DKK1 protein (for example, in the case of human, NCBI accession number: NM_012242), sclerostin (for example, in the case of human, NCBI accession number: NM_025237), IWR-1 ( Merck Millipore), IWP-2 (Sigma-Aldrich), IWP-3 (Sigma-Aldrich), IWP-4 (Sigma-Aldrich), IWP-L6 (EMD Millipore), C59 (or Wnt-C59) (Cellagen technology) ), ICG-001 (Cellagen Technology), LGK-974 (or NVP-LGK-974) (Cellagen Technology), FH535 (Sigma-Aldrich), WIKI4 (Sigma-Aldrich), KYO2111 (Minami I, et al, Cell) Rep. 2: 1448-1460, 2012), PNU-74654 (Sigma-Aldrich), XAV939 (Stemgent), and derivatives thereof. In the production of cerebral cortical neurons from the pluripotent stem cells of the present invention, a preferred Wnt inhibitor is a substance that suppresses Wnt production, and for example, PORCN (in the case of human, the accession of NCBI is involved in the processing of Wnt protein). No.: NP_001269096, NP_073736, NP_982299, NP_982300, or NP_982301) are exemplified, and C59, IWP-3, IWP-4, IWP-L6 or LGK-974 are preferred. , C59 are more preferred.

The concentration of C59 in the culture medium is, for example, 0.1nM, 0.5nM, 1nM, 2nM, 2.5nM, 3nM, 4nM, 5nM, 6nM, 7nM, 7.5nM, 8nM, 9nM, 10nM, 20nM, 30nM, 40nM, 50nM. , 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, but not limited thereto. The concentration is preferably 1 nM to 50 nM, for example, 2 nM to 50 nM, more preferably 10 nM to 50 nM, or a concentration of less than 10 nM, for example, 2 nM or more and less than 10 nM.

In the present invention, LGK-974 can also be preferably used as a Wnt inhibitor. The concentration of LGK-974 in the culture medium is not particularly limited as long as it is a concentration that inhibits Wnt, for example, 1nM, 10nM, 25nM, 50nM, 100nM, 150nM, 200nM, 500nM, 750nM, 1μM, but these Not limited. The concentration is preferably 1 nM to 1 μM, for example, 1 nM to 500 nM, more preferably 10 nM to 200 nM, or a concentration of 10 nM to 150 nM, for example, 10 nM or more and 100 nM or less.

<BMP inhibitor>
The “BMP inhibitor” is as described above, and examples of the BMP inhibitor include the same as those described in the method for producing dopamine neural progenitor cells. The BMP inhibitor used in this step is preferably LDN193189.

The concentration of LDN193189 in the culture medium is, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM. , 40 μM, 50 μM, but not limited thereto. The concentration is preferably 2 μM or less, for example, 100 nM to 2 μM, more preferably 500 nM to 2 μM, or a concentration of less than 2 μM, for example, 100 nM or more, less than 2 μM, and more preferably Is 500 nM or more and less than 2 μM.

<Process (i)>
The culture medium used in step (i) can be prepared by using the medium used for culturing animal cells as a basal medium and adding the above-mentioned TGFβ inhibitor, bFGF, Wnt inhibitor, and BMP inhibitor. Examples of the basal medium include the same as those described in step (i) of the method for producing dopaminergic neural progenitor cells. The medium is preferably DMEM or a mixture of DMEM and Ham's F12 medium at a ratio of 1:1. Serum may be contained in the basal medium, or a serum substitute may be added instead of serum. Serum substitutes include, for example, albumin, transferrin, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, and multiple combinations selected from these. Are listed. Preferably, the serum replacement is KSR.

When KSR is used in the step (i), the concentration in the basal medium is, for example, 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%. %, 20%, but preferably less than 20%, for example 10% or more and 15% or less. The basal medium contains 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low molecular compounds, antibiotics, antioxidants, and pyruvin. It may also contain one or more substances such as acids, buffers, inorganic salts and the like. A preferred basal medium is DMEM containing KSR, 2-mercaptoethanol, pyruvic acid, non-essential amino acids and L-glutamine, N2 supplement and/or B27 supplement, or a medium in which Ham's F12 medium is mixed 1:1. ..

In step (i), the pluripotent stem cells may be dissociated and used, and examples of the method for dissociating the cells include a method of mechanically dissociating, a dissociation solution having a protease activity and a collagenase activity (for example, Accutase ( (Trademark), Accumax (trademark) or the like) or a dissociation method using a dissociation solution having only collagenase activity. Preferably, Accumax is exemplified) to dissociate pluripotent stem cells is used. When the cells are dissociated, a ROCK inhibitor may be appropriately added after the dissociation and cultured.

The culture in step (i) is preferably performed by the above-mentioned suspension culture. The culture conditions (culture temperature, CO ₂ concentration, O ₂ concentration) in step (i) are the same as the culture conditions in step (i) described in the method for producing dopamine neural progenitor cells.

The number of days of the step (i) of the present invention is not particularly limited, and examples thereof include 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, or more days, and 36 days or less. , 30 days or less, 24 days or less, 18 days or less, 12 days or less. It is more preferably 3 to 12 days, still more preferably 6 days.

<Step (ii)>
As the culture solution used in the step (ii) of the present invention, a basal medium used for culturing animal cells can be used. Examples of the basal medium include the same as those described in step (i) of the method for producing dopaminergic neural progenitor cells. Preferred is a medium in which DMEM and Ham's F12 medium are mixed at a ratio of 1:1. Serum may be contained in the basal medium, or a serum substitute may be added instead of serum. Examples of the serum substitute include albumin, transferrin, KSR, N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen precursor, trace elements, and a plurality of combinations thereof. Preferably, the serum replacement is N2 supplement and B27 supplement. The basal medium contains 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low molecular compounds, antibiotics, antioxidants, and pyruvin. It may also contain one or more substances such as acids, buffers, inorganic salts and the like. The culture medium used in step (ii) is, for example, FGF8 (fibroblast growth factor 8), SHH signal inhibitor (eg: Cyclopamine, Forskolin, Rapamycin (mixture of isomers), etc.) in the above basal medium. It is possible to use those to which a neurotrophic factor or the like is appropriately added.

In the present invention, FGF8 is not particularly limited, but in the case of human FGF8, four splicing forms of FGF8A, FGF8b, FGF8e or FGF8f are exemplified, and in the present invention, FGF8b is more preferable. FGF8 is commercially available from, for example, Wako or R&D systems, and can be easily used, but it may be obtained by forced expression in cells by a method known to those skilled in the art.

The concentration of FGF8 in the culture medium is, for example, 1 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 150 ng/ml, 200 ng/ml, 250 ng/ml, 500 ng/ml, 1000 ng/ml. , 2000 ng/ml, 5000 ng/ml, but not limited to these. It is preferably 50 ng/ml.

The step (ii) is a step of culturing the cells obtained in the step (i), and the culture may be adherent culture or suspension culture. When adherent culture is performed, it can be performed by culturing the extracellular matrix in a culture vessel coated with the extracellular matrix. The coating treatment can be performed by placing a solution containing an extracellular matrix in a culture vessel and then removing the solution as appropriate.

The “extracellular matrix” is as described above, and is preferably a mixture of polyornithine, laminin and fibronectin.

The culture conditions (culture temperature, CO ₂ concentration, O ₂ concentration) of step (ii) are the same as the culture conditions of step (i) described in the method for producing dopamine neural progenitor cells.

The culture period of step (ii) is, for example, preferably 1 day or more, 2 days or more, 3 days or more, 4 days or more, and preferably 20 days or less, 15 days or less, 10 days or less. More preferably, it is 5 to 7 days.

A method known per se can be used for the isolation of neural progenitor cells from the living body. As such a method, in the case of cerebral cortical neural progenitor cells, for example, as described in Non-Patent Document 6 or as shown in Examples, dorsal pallium is cut out from the cerebrum of a mouse fetus, and a nerve cell dispersion liquid is used. Examples of the method include culturing the cells after the dispersion.

3. Kit for selecting neural progenitor cells The present invention also provides a kit for selecting neural progenitor cells from pluripotent stem cells. This kit contains, in addition to the above-mentioned miRNA-responsive mRNA and DNA encoding the mRNA, the transfection control mRNA, the DNA encoding the mRNA, and a nerve containing a specific factor for inducing neural progenitor cells. The progenitor cell inducer (eg, freeze-dried product, frozen solution dissolved in an appropriate buffer, etc.), pluripotent stem cells, reagents and culture medium may be contained. It may include written documents and instructions. Further, it may contain an antibody for selection using a cell sorter or a drug such as the above-mentioned antibiotic for selection without using a cell sorter. Preferably, the drug is puromycin.

4. Drug containing neural progenitor cells The present invention provides a drug containing neural progenitor cells. Here, the neural progenitor cells are not particularly limited as long as they are cells obtained by the method of the present invention. The neural progenitor cells are used as they are or as cell masses such as pellets concentrated by filtration with a filter. Furthermore, the drug can be cryopreserved by adding a protective agent such as DMSO (dimethyl sulfoxide). For safer use as a medicine, the medicine is subjected to treatment such as heat treatment and radiation treatment under conditions such that the protein of the pathogen is denatured while leaving the function as a target neural progenitor cell. May be. In addition, in order to prevent the neural progenitor cells from growing more than necessary, in combination with the above treatment, suppression of proliferation by mitomycin C pretreatment or the like, or a gene of a metabolic enzyme that mammals do not naturally have is concerned. It is introduced into cells, and then an inactive drug is administered if necessary, and the drug is transformed into a toxic substance only in cells into which genes for metabolic enzymes not naturally possessed by mammals have been introduced. May be subjected to a treatment such as a method of killing syrup (suicide gene therapy).

The medicament of the present invention can be administered to mammals (eg, mouse, rat, hamster, guinea pig, dog, monkey, orangutan, chimpanzee, human, etc.). Examples of the administration form (transplantation method) of the drug to humans include the methods described in Nature Neuroscience, 2, 1137 (1999) or N Engl J Med.;344:710-9 (2001). To be Preferably, the medicament of the present invention is administered (transplanted) to a dopamine-deficient region of the brain. In the medicament of the present invention, it is preferable to use neural progenitor cells prepared using cells of the patient himself or cells of a donor whose histocompatibility is within an acceptable range, but sufficient cells can be obtained for reasons such as age and constitution. If not, it can be embedded in a capsule such as polyethylene glycol or silicone, a porous container, or the like and transplanted while avoiding the rejection reaction. Further, the dose (transplantation amount) and the number of administrations (transplantation number) of the drug of the present invention can be appropriately determined depending on the age, body weight, symptoms, etc. of the patient to be administered.

The drug containing the neural progenitor cells of the present invention can be efficiently engrafted in the patient's body by administration (transplant) of itself. Therefore, when the medicament of the present invention contains dopamine neural progenitor cells, diseases caused by decreased production (release) of dopamine, for example, neurodegeneration such as Parkinson's disease, Huntington's disease, Alzheimer's disease, epilepsy and schizophrenia. Useful for treating diseases. When it contains cerebral cortex neural progenitor cells, it is useful for treating, for example, patients with Alzheimer's disease, Pick's disease, progressive myoclonus epilepsy, etc., or patients with serious damage to the cerebral cortex due to accidents or cerebral infarction.

The contents of all publications, including patents and patent applications, cited herein are hereby incorporated by reference to the same extent as if all were expressly incorporated by reference. It is a thing.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the examples shown below.

In the examples described below, experiments were conducted as follows.
<Culturing of human iPS cells>
Feeder-free human iPS cells (1147F1 and 1231A3 strains) were subjected to iMatrix-511 (E8) (S8) in StemFit (+bGF) medium according to a previously reported protocol (Nakagawa et al. (2014) Sci Rep. 4:3594) ( Nippi) maintained on. Human iPS cells were passaged every 8 days. Prior to passage, 6-well plates were coated with iMatrix-511 (E8) diluted to a density of 0.5 μg/cm ² with sterile PBS for at least 1 hour at 37°C. After aspirating the PBS, it was immediately replaced with 1.5 ml StemFit containing 10 μM ROCK inhibitor (Y-26732). To harvest human iPS cells, old media was aspirated and cells were washed with 1 ml PBS. After removing the PBS by suction, 0.3 ml of TrypSELECT solution (see online protocol) was added to each well, and the plate was transferred to an incubator. Then, TrypSELECT medium was aspirated and washed with 1 ml of PBS. The PBS was aspirated, and 1.5 ml of StemFit (containing a ROCK inhibitor) was added to each well. Cells were harvested from the matrix using a rubber end cell scraper and pipetted into single cells. Cell densities were calculated by trypan blue staining and Cell Countess (Invitrogen), seeded at 1.3×10 ⁴ cells/well and mixed rapidly to evenly disperse the cells. After 24 hours, the whole amount was replaced with StemFit (without ROCK inhibitor), and then the medium was replaced every other day.

<ES cell culture>
Human ES cells (Kh1 strain) and mouse ES cells were cultured by the same method as the above iPS cells.

<Method for inducing differentiation into dopaminergic neural progenitor cells>
From human iPS cells (1147F1 and 1231A3 strains) and human ES cells (Kh1 strain), dopamine neural progenitor cells were induced to differentiate by the following method. A 6-well plate was coated with iMatrix-511 (E8) in the same manner as above, and 4 ml/day of differentiation medium (d0 differentiation medium: 100 μM sodium pyruvate, 100 μM β-mercaptoethanol (2ME) was added. Glasgow's MEM/8% knockout-serum (KSR) (8GMK medium), 10 μM Y-26732 (Wako), 100 nM LDN-193189 (Stemgent), 500 nM A83-01 (Wako) were added. Differentiation was initiated from a dispersed cell suspension of human iPS in d0 differentiation medium (with ROCK inhibitor) and seeded in 6 well plates at 5×10 ⁶ cells/well. The plate was gently agitated and then returned to the incubator. After 24 hours, the entire medium was replaced with d1 medium or the like (see the composition below), and the medium was replaced daily. Per well, 4 ml from 0 to 2 days, 6 ml from 3 to 6 days, 8 ml from 7 to 8 days and 10 ml from 9 to 11 days were added. The number of cells increased to about 1×10 ⁷ cells per well in the first 11 days of culture. Cells were passaged on days 12, 20 and 29 and were always seeded in 6 well plates at 5x10 ⁶ cells/well. From day 12 onwards cells were always maintained in Neurobasal B27 medium supplemented with: 8 ml of medium was added per well and replaced.
On the 1st and 2nd days, a medium in which 100 nM LDN-193189, 500 nM A83-01, 2 μM purmorphamine (Calbiochem) and 100 ng/ml human recombinant FGF-8 (Wako) were added to 8GMK medium was used. From day 3 to 7, add 8 nGMK medium containing 100 nM LDN-193189, 500 nM A83-01, 2 μM purmorphamine, 100 ng/ml human recombinant FGF-8 and 30 μM CHIR99021 (Stemgent). did. On the 7th to 11th days, a medium prepared by adding 100 nM LDN-193189 and 30 μM CHIR99021 to 8GMK medium was added. From day 12 onward, Neurobasal B27 medium was supplemented with 200 μM ascorbic acid (Sigma Aldrich), 400 μM dbcAMP, 20 ng/ml human recombinant BDNF and 10 ng/ml human recombinant GDNF.
On days 12, 20 and 29, mixed cells were evaluated for differentiation efficiency by staining with Kan Research anti-CORIN (bottom plate marker) antibody and PSA-NCAM antibody labeling the nervous system. In parallel, approximately 5×10 ⁵ cells in 200 μl staining buffer (including ROCK inhibitor) were diluted 200-fold with mouse anti-human CORIN antibody (Kan Research) or 200-fold diluted with anti-human PSA-NCAM mouse monoclonal. Stained with any of the antibodies (MAB5324, Millipore). Alexa 488-conjugated anti-mouse IgG/IgM antibody was used at 400-fold dilution for CORIN and PSA-NCAM, respectively. Cells were stained at 4°C for 30 minutes. The cells were washed twice with HBSS medium (Hank's balanced salt solution/2% KSR, 50 μg/ml penicillin/streptomycin and 10 μM Y-26732). Dead cells were stained with 7-AAD for 10 minutes before FACS analysis.
Cells were analyzed on either BD FACS aria-II or BD Accuri using standard filters for FITC and 7-AAD signals. Gates were set to exclude dead cells based on SSC-A intensity (P1) and remove doublets from FSC-W vs. FSC-H (P2) and SSC-W vs. SSC-H (P3). Finally, cells stained with 7-AAD were excluded, leaving the "LIVE" cell gate (P4/LIVE). 20,000 cells gated with P4 were analyzed. CORIN positive and PSA-NCAM positive gates were set up to have 0.2% or less cells in Alexa 488-IgG or IgG isotype control secondary antibody.

<Method for inducing differentiation into forebrain neural progenitor cells>
From human iPS cells (1231A3 strain) and human ES cells (Kh1 strain), except that purmorphamine, FGF8 and CHIR were not added to the medium, the same as the above dopamine neural progenitor cells until the 12th day from the initiation of differentiation induction. Differentiation was induced by the method.

<Method for inducing differentiation into cortical neurons>
Mouse ES cells maintained on feeder cells were washed twice with PBS buffer after removing the medium, treated with 0.05% trypsin EDTA for 5 minutes at 37° C., and collected by centrifugation. Colonies were collected in an en block and left in a 10 cm dish coated with 0.01% gelatin coat for 30 minutes to remove medium components. Further, the supernatant was removed by centrifugation, and washed twice with PBS buffer. 1 ml of Accumax (Funakoshi) was added and treated at 37°C for 5 minutes. After making a single cell by pipetting, 10 ml of PBS buffer was added and the mixture was centrifuged. After removing the supernatant, the number of cells was counted and seeded in a 96-well U-bottom dish at 4,000 cells per well.
On days 0 to 6, GMEM (Gibco) medium supplemented with NEAA, L-glutamic acid, 0.1 mM 2ME, 1 mM pyruvic acid and 10% KSR was used and cultured at 37° C. in an atmosphere of 5% CO ₂ . The medium was changed every 3 days. 20 nM C59 and 10 μM SB431542 were added to the medium at the final concentration at the start of the culture, and were similarly added when the medium was exchanged after 3 days.
Subcultured on the 6th day from the start of culture, 500 ml of DMEM (Gibco) supplemented with 2ME and L-glutamic acid, 5 ml of N2 supplement and 10 ml of B27 were used, and 37°C, 5% CO ₂ was used. Incubated under atmosphere. On the 8th day from the start of culture, rmFGF8b (final concentration 50 ng/ml) and cyclopamine (final concentration 5 μM) were added, and the cells were cultured at 37° C. in an atmosphere of 5% CO ₂ /40% O ₂ .

<Preparation of DNA construct for preparation of miRNA-responsive mRNA>
The DNA construct contains a 5'UTR cassette, a marker protein ORF, and a 3'UTR cassette from the 5'side. Most of the DNA constructs used in this experiment were designed so that the miRNA target sequence was inserted into the 5'UTR cassette and the translation of the marker protein was suppressed when the miRNA bound to this target sequence. The oligos and primers used are shown in Table 1, and the PCR conditions are shown in Tables 2-4.

Untranslated Region The 5'UTR-encoding oligo optimized for efficient translation in mammalian cell lines was used as a template to generate a 5'UTR PCR product (see Table 1). This product contains 5'guanosine that is capped after in vitro transcription and the T7 promoter used for T7 RNA polymerase in in vitro synthesis. Furthermore, a 3'UTR PCR product was prepared using the optimized 3'UTR as an template and an oligo containing a terminal 20 nt thymidine repeat as a template. Both 5'UTR and 3'UTR products were generated using a 2-step PCR program (see Tables 3 and 4).

Open reading frame (ORF)
The ORF was amplified from the plasmid using primers that were partially complementary to the 3'end of the 5'UTR PCR product and the 5'end of the 3'UTR PCR product. See Table 2 for PCR details. All ORFs were made using a 2-step PCR program (see Tables 3 and 4). The sequences of the obtained puromycin resistance gene, tagBFP and ORF of EGFP are shown in SEQ ID NOs: 745, 747 and 749, respectively.

Fusion PCR
From a fusion PCR of a 3'UTR extended with a reverse primer (3UTR120A) containing a 5'untranslated region (5'UTR), ORF and an additional 100-nt adenosine tail (totaling 120 polyA tails) , A full-length DNA template (see Figure 2b) was prepared. All fusion PCRs were performed using a 3-step PCR program (see Tables 3 and 4).

A template for miRNA-responsive mRNA was prepared by using the above-mentioned PCR product and an oligo containing the target sequence of miRNA (see FIGS. 2c and 2d). In addition, SEQ ID NOS: 1 to 642 show the sequences of 5'UTR containing 642 types of miRNA target sequences (Table 8).

<in vitro transcription (IVT)>
MRNA synthesis was performed using the MEGAscript T7 kit (Ambion) and the DNA construct prepared above. In this reaction, pseudouridine-5'-triphosphate and methylcytidine-5'-triphosphate (TriLink Bio Technologies) were used instead of uridine triphosphate and cytidine triphosphate, respectively. Prior to the IVT (mRNA synthesis) reaction, guanidine-5'-triphosphate was diluted 5-fold with Anti Reverse Cap Analog (New England Biolabs). The reaction mixture was incubated at 37°C for 5 hours, TURBO DNase (Amibion) was added, and the mixture was further incubated at 37°C for 30 minutes. The obtained mRNA was purified with FavorPrep Blood/Cultured Cells total RNA extraction column (Favorgen Biotech) and incubated at 37° C. for 30 minutes using Antarctic phosphatase (New England Biolabs). Then, it was further purified by RNeasy Mini Elute Cleanup Kit (QIAGEN). The sequences of the obtained puromycin resistance gene mRNA for control, tagBFP mRNA and EGFP mRNA are shown in SEQ ID NOs: 753 to 755, respectively.
Hereinafter, the mRNA produced by IVT from the DNA construct is referred to as miRNA switch. For example, a miRNA switch prepared from "miR-9-5p-tagBFP" is described as "miR-9-5 switch". Regarding the tandem switch, the miRNA switch made from "1x9-5p-tagBFP-4x9-5p" is the miRNA switch made from "tandem miR-9-5p switch" and "1x9-5p-BimEL-4x9-5p" below. Is described as "Bim switch".

<Introduction of miRNA switch>
Introduction of miRNA switch was performed according to the schedule shown in FIG. 1b.
On the first day of transfection, differentiation-induced neural progenitor cells (adherent culture) were incubated with Accumax for 10 minutes to collect detached cells, and the number of cells was counted with Countess. A cell suspension was prepared by diluting with a NBB27+GABA+Y medium so that the cell concentration was 5.0×10 ⁵ cells/ml. 1 ml of cell suspension (500,000 cells/well) was gently dispensed into a 24-well plate (iMatrix coated). It was cultured overnight in an incubator at 37° C. and 5% CO ₂ .
An mRNA (hereinafter referred to as a transfection control) encoding a fluorescent protein gene different from the miRNA-responsive mRNA was introduced into each cell together with the miRNA switch.
When mRNA was introduced without seeding cells, 12.5 μl of Stemfect RNA transfection kit (Stemgent) was added to each of two sterile 1.5 ml tubes. In the first tube (A), 1.0 μl of Stemfect RNA transfection reagent was added to the buffer and mixed. In the second tube (B), 1.0 ng of 100 ng/μl miRNA switch and 1.0 μl of 100 ng/μl EGFP mRNA were added to the buffer and mixed. Solution A and solution B were mixed and incubated at room temperature for 15 minutes. Meanwhile, the medium was replaced with 500 μl of fresh I ₃ medium without antibiotics. After 15 minutes, the mixture was added to the cells, the plate gently shaken, and incubated at 37° C. for 4 hours in a 5% CO ₂ atmosphere. After 4 hours, the medium was aspirated, the well was washed once with IMDM, and 500 μl of fresh I ₃ medium was added to the well. After 24 hours, Accumax was added and incubated for 10 minutes, and then cells were collected and analyzed or sorted using FACS.
When seeding cells and introducing mRNA, add 200 μl of Accumax to cells cultured in 24-well plate, incubate at 37°C, 5% CO ₂ for 10 to 15 minutes, and then add 500 μl of medium. (DMEM 2%HS) was added, and the mixture was collected in a 15 ml tube and centrifuged at 4° C., 2000 rpm, 10 minutes to remove the supernatant. After adding 500 μl of medium and pipetting, the number of cells was counted by cell countess (invitrogen), and diluted as needed. When reseeding to a 24-well plate, cells were reseeded at 2×10 ⁵ cells/well, and when reseeding to a 6-well plate, cells were reseeded at 1×10 ⁶ cells/well. At the same time when the cells were seeded in the well, the mixed solution containing the mRNA prepared by the above method was added to the well. Incubated at 37° C., 5% CO ₂ for 4 hours. Then, the medium was removed, and the required amount of medium (DMEM 2%HS) was added to the wells. After 24 hours, it was analyzed using FACS.

<Search for miRNA switches that can be used for selection of dopaminergic neural progenitor cells>
Using dopamine neural progenitor cells differentiated from human iPS cells (1147F1 strain and 1231A3 strain), FACS selection was performed on the 13th day of differentiation using an antibody against CORIN which is a bottom plate marker protein. RNA was extracted from CORIN-positive cells (dopaminergic neural progenitor cells) and negative cells (other than dopaminergic neurons) and subjected to miRNA array. Performed using Agilent Technologies Human miRNA Microarray Release 19.0 and SurePrint G3 Human GE Microarray as per manufacturer's instructions. Data were analyzed using GeneSpring GX12.6 software (Agilent Technologies). MiRNAs that are more highly expressed in dopaminergic neural progenitor cells at a rate of change of 2 or more in each strain, and miRNAs that overlap in 2 strains or published literature (eg, Huang T, J Mol Cell Biol 2: 3, 152-63), the miRNA switch was prepared from the target sequences of 37 types of miRNAs, including miR-9-5p and miR-218-5p, by using the above-mentioned method as a candidate.
The cells introduced with these miRNA switches were analyzed by FACS to determine whether they function as miRNA switches.

<Analysis by FACS>
A 24-well plate, 200 μl of Accumax per well was added to each cell to be analyzed (1 ml for a 6-well plate), and the cells were incubated at 37° C. under 5% CO ₂ for 10 to 15 minutes. After that, 500 μl of medium (DMEM 2%HS) was added, the cells were collected in a 15 ml tube, and centrifuged at 4° C., 2000 rpm, 10 minutes, and the supernatant was removed. 200 μl of medium (DMEM 2%HS) or HBSS was added per well, suspended by pipetting, passed through a filter and used for analysis.

<Time-dependent analysis of miR-9-5p and miR-218-5p expression levels in dopamine neural progenitor cells>
Human iPS cells (1231A3 strain) and human ES cells (Kh1 strain) are induced to differentiate into forebrain or dopaminergic neural progenitor cells, respectively, and the cells are collected every 7 days from 0 to 35 days after the initiation of differentiation induction, and the liquid is collected. After freezing with nitrogen, it was stored at -80°C. For RNA extraction, cell pellets were thawed on ice and resuspended in Trizol reagent (Invitrogen) according to the manufacturer's protocol. RNA was precipitated in ice-cold isopropanol overnight in the freezer, washed with 75% ethanol, dried, and finally resuspended in an appropriate volume of RNase-free purified water and heated at 50°C for 5 minutes to suspend dried RNA. Aided turbidity. Following the manufacturer's protocol, the residual genomic DNA was digested using the TURBO DNase (Ambion) kit and the slurries were used to remove post-reaction enzymes and excess cations. RNA purity and quantity was initially measured with a Nanodrop 2000 (ThermoFisher). Specific for hsa-miR-9-5p (miRBase.v21: MIMAT0000441; Assay ID. 000583, Catalog No. 4427975) and hsa-miR-218-5p (miRBase.v21: MIMAT0000275; Assay ID. 000521, Catalog No. 4427975) 10 ng of total RNA was used for reverse transcription according to the manufacturer's protocol of the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) using different RT primers. Use 1.33 μl reverse transcription reaction with nuclease-free purified water along with 1xTaqMan® Universal PCR Master Mix II (without UNG) and 1xTaqMan® Small RNA Assays as directed for the small RNA TaqMan Assay. It was set to 20 μl. PCR was performed using the StepOnePlus ^™ real-time system (Applied Biosystems) under standard temperature gradient conditions. To compare the expression of mature miRNA with the state of iPS cells on day 0 of differentiation, the expression level was standardized using Universal RNU6B TaqMan® Small RNA Assay (NR_002752; Assay ID. 001093, Catalog No. 4427975). (FIG. 4) and analyzed by the -ΔΔCt method.

<Expression analysis of various marker genes using qRT-PCR in cells selected by miRNA switch>
Total RNA was extracted from FACS sorted cells using the miRNA switch or cells sorted with the Bim switch. Reverse transcription reaction was performed using ReverTra Ace kit (TOYOBO) and oligo(dT)20 primer.
For quantitative PCR, expression analysis of various marker genes was performed using TaqMan probe and StepOne Plus Real-Time PCR System (Applied Biosystems). Table 5 shows the primers for human genes and Table 6 shows the primers for mouse genes used for the analysis.

<FACS selection using miRNA switch>
Human iPS cells (1231A3 strain) are induced to differentiate into dopamine neural progenitor cells, and 14 days, 21 days and 28 days after the initiation of differentiation induction, miR-9-5p switch or miR-218-5p switch is introduced, The next day, it was sorted by FACS (Fig. 3). RNA was extracted from the selected cells, and the expression level of each marker gene was analyzed by qRT-PCR according to the method described above. The expression level of each marker gene was compared between miRNA switch positive cells and negative cells (FIGS. 5 to 7). In addition, the expression of the marker protein was examined by immunostaining by the following method (Fig. 9).

<Comparative experiment: FACS sorting of dopamine neural progenitor cells using anti-CORIN antibody>
Human iPS cells (1231A3 strain) were induced to differentiate into dopaminergic neural progenitor cells, and cells at 12 days after the initiation of differentiation were added to 24-well plates, 200 μl of Accumax per well, and incubated at 37°C, 5% CO ₂ for 10 days. Incubated for ~15 minutes. Thereafter, 500 μl of medium (DMEM 2% HS) was added, the cells were collected in a 15 ml tube, and centrifuged at 4° C., 2000 rpm, 10 minutes, and the supernatant was removed. 200 μl of medium (DMEM 2%HS) or HBSS was added per well, suspended by pipetting, passed through a filter and used for analysis. Antibodies used in the analysis were anti-CORIN mouse IgG antibody (1:200, transferred from KAN Research Institute) and Alexa 488-conjugated anti-mouse IgG antibody (1:400, Invitrogen). The antibody was added and incubated at 4°C for 20 minutes, and the cells were washed twice with HBSS buffer. Dead cells were discriminated by 7-AAD staining. The standard for positive staining was set so that 0.1% or less of the samples exceeded the threshold of the sample that lost the primary antibody. BD FACS Aria III or BD LSR Fortess was used for analysis, and BD FACS Aria III was used for selection. RNA was extracted from a sample obtained by continuously culturing the selected cells on day 21 or day 28, and the expression level of each marker gene was analyzed by qRT-PCR according to the above method (FIG. 8).

<Immunostaining>
For immunostaining of FACS-sorted cells, after FACS sorting, a cell aggregate was used by continuing culturing on a 96-well low-adhesion plate for 8 days.
For immunostaining of cells sorted by Bim switch, after sorting, cells were further cultured on a 96-well low-adhesion plate for 7 days to form cell clumps, which were then dissociated into single cells by Accumax, and 8 well chamber slide ( iMatrix coat) and the cells after 7 days were used as a sample.
These cells were fixed with 4% PFA and suspended in PBS buffer (Sigma) supplemented with 2% goat serum and 0.5% saponin, and each cell was stained with a primary antibody specific for each marker protein. Alexa 488-conjugated anti-mouse IgG antibody (Millipore) was diluted 500 times and used as a secondary antibody. Nuclei were stained with Hoechst (Invitrogen) diluted 10,000 times. Stained images were taken with a Biorevo BZ-9000 microscope (Keyence). The antibodies used in this experiment are shown in Table 7.

<Additional culture>
A part of the positive or negative cells collected by FACS and collected was used as an RNA extraction sample, and the others were further dispersed in a 96-well low-adhesion plate (Sumitomo Bakelite, PrimeSurface), and a week of culture was added to aggregate clumps ( (also referred to as aggregation or sphere), and used as a sample for RNA extraction, or after fixation with 4% PFA, a frozen section was used as a sample for immunostaining.

<FACS selection using tandem miRNA switch>
Human iPS cells (1231A3 strain) were induced to differentiate into dopamine neural progenitor cells, and miR-9-5p switch or tandem miR-9-5p switch was introduced 21 days after the initiation of differentiation induction, and FACS selection was performed by the above method. (Figs. 10, 11).

<Transfection of Bim switch>
Bim switch transfection was performed according to the schedule shown in FIG. 1b on the 21st day of differentiation induction by the following method.
For one well of a 6-well plate, 50 μl of Stemfect transfection buffer was added to each of two sterile 1.5 ml tubes. In the first tube (A), 4.0 μl of Stemfect RNA transfection reagent was added. In the second tube (B), 100 ng/μl of miR-Bim switch was added at 1.0, 1.5 or 2.0 μl and 100 ng/μl of puromycin resistant mRNA at 2.0 μl. Solution A and solution B were mixed and incubated at room temperature for 15 minutes. Meanwhile, the medium was replaced with 2 ml of fresh I ₃ medium without antibiotics. After 15 minutes, the mixture was added to the cells, the plate gently shaken, and incubated at 37° C. for 4 hours in a 5% CO ₂ atmosphere. After 4 hours, the medium was removed, the wells were washed once with IMDM, and 2 ml of fresh I ₃ medium containing 2 μg/ml puromycin was added to the cells.
The next day, surviving cells were collected and seeded on a low-adhesion 96-well plate at 2×10 ⁴ cells/well to prepare an aggregation, and additional culture was performed for 7 days. On the 28th day of induction of differentiation, aggregation was collected, and a part of the collected aggregation was subjected to qPCR for expression analysis of each marker gene. In addition, 1 ml of Accumax was added to a part of the remaining aggregation and treated at 37° C. for 5 minutes. After forming a single cell by pipetting, it was seeded on an 8-well chamber slide (iMatrix coated), and after 7 days (the 35th day of differentiation induction), fixed with 4% PFA and immunostained in the same manner as above.

<Optimization of puromycin selection>
Human ES cells (Kh1 strain) and human iPS cells (1231A3 strain) were differentiated into neural progenitor cells by the method described above, and the examination was carried out 20 and 27 days after differentiation. 96 well plate (flat bottom, iMatrix coated) was seeded at 1.7 × 10 ⁵ cells/well, and the next day, the medium was replaced with puromycin at a final concentration of 0 to 15 μg/ml and cultured for 20 hours. The cell survival rate was evaluated by the Diret Cell Proliferation Assay Kit (Invitrogen) (Fig. 12a). As a control, autofluorescence of cells not treated with CyQuant kit was measured.
Then, puromycin-containing mRNA was added to a medium to a final concentration of 10 μg/ml at a concentration of 0 to 100 ng/ml to purify each cell, and the cells were cultured for 20 hours. The residual rate was evaluated (Fig. 12b).
Puromycin resistant mRNA was transfected to cells at 100 ng/ml, and mRNA used as a Bim switch was further transfected at 0 to 100 ng/ml, and puromycin was added to a final concentration of 10 μg/ml. After culturing in the medium for 20 hours, the cell survival rate was evaluated in the same manner as above, and the cell state and cell recovery rate were examined (FIGS. 12c and 13). In addition, for comparison, cells in FACS selection were also examined for cell state and cell recovery rate (FIG. 13b). Collection efficiency (%) is calculated by the number of cells recovered/the number of cells plated before transfection. At this time, the BFP positive cells by FACS were 10 to 30%, and the positive cells are sorted by FACS.
Finally, RNA was extracted from the surviving cells sorted by Bim switch, and the expression level of each marker gene was analyzed by qRT-PCR by the same method as described above (Fig. 14) to show the expression status of each marker protein. Confirmed by immunostaining (Fig. 15).

<Selection by miRNA switch in cortical neural progenitor cells derived from mouse ES cells>
Cerebral cortical neural progenitor cells were induced to differentiate from mouse ES cells and FACS-sorted by the method described above using the miR-9-5p switch (Fig. 16a). In addition, RNA was extracted from sorted cells (miR-9-5p positive or negative) and unsorted cells, and the expression of each marker gene was analyzed by qRT-PCR by the same method as above (FIGS. 16b to d). ).

<Screening of miRNA that can be used for selection of dopaminergic progenitor cells>
Introducing 642 types of miRNA switches (miRNA switches containing the sequences of SEQ ID NOs: 1 to 642) into dopamine neural progenitor cells differentiated from human iPS cells, the responsiveness to miRNAs expressed in dopamine neural progenitor cells was determined by FACS. Was analyzed using.

<Screening of miRNA that can be used for selection of cortical neural progenitor cells>
A 13-day-old pregnant mouse was euthanized with Somnopentyl, and the fetus was removed as much as possible. The cerebrum was excised from the excised E13.5 mouse, the dura was peeled off, and the dorsal pallium was excised and collected in the state where only nerve cells were present. All treatments were performed in ice-cold HESS and completed within 30 minutes. The collected dorsal pallium was dispersed using a nerve cell dispersion liquid (DS Pharma biomedical, MB-X9901D), and seeded in a flat bottom dish coated with PDL at a density of 15×10 ⁴ cells/cm ² . At the same time, 642 kinds of miRNA switches were introduced into cells by the same method as above. Twenty-four hours later, the responsiveness to miRNA expressed in cerebral cortical neural progenitor cells was analyzed using FACS by the same method as described above.

Example 1: Search for miRNA that can be used for selection of dopamine neural progenitor cells As a result of FACS analysis performed using differentiation-induced dopamine neural progenitor cells, in the case of using miR-9-5p switch and miR-218-5p In addition, it was demonstrated that transfection with miRNA switch enables good selection of dopaminergic neural progenitor cells 21 and 28 days after the initiation of differentiation induction (FIG. 3). From this result, it was determined that, in the selection of dopaminergic neural progenitor cells, the miRNA switch is transfected 21 days after the initiation of differentiation induction.

Example 2: Time-dependent analysis of miR-9-5p and miR-218-5p expression in midbrain and forebrain nerve cells Human iPS cells (1231A3 strain) and human ES cells (Kh1 strain) were forebrain or midbrain, respectively. And the expression levels of miR-9-5p and miR-218-5p were examined over time by qRT-PCR. The expression level was standardized using the results of RNU6B. As a result, miR-9-5p has a low expression level in the 1231A3 strain on the 14th day from the initiation of differentiation induction, and in any of the strains from the 21st day onward, any of the cases differentiated into the forebrain or midbrain, Expression was seen. However, on day 28 and 35, the expression level was maintained high when differentiated into the midbrain in all cell lines, but the expression level decreased when differentiated into the forebrain. I was seen. From this, it was suggested that miR-9-5p is particularly suitable for selection of the midbrain nerve, and that it is suitable to perform selection after the 21st day (Fig. 4). On the other hand, for miR-218-5p, after 7 days from the initiation of differentiation induction, expression was confirmed in any case where it was differentiated into any strain and forebrain or midbrain, and when differentiated into midbrain Although there was a tendency that the expression level was higher than that in the case of being differentiated into the forebrain, it was not a significant difference (Fig. 4).

Example 3: FACS selection of dopamine neural progenitor cells using miRNA switch Dopamine neural progenitor cells differentiated from human iPS cells were transfected with miR-9-5p switch or miR-218-5p switch on day 21. Then, FACS selection was performed to examine the expression level of each marker gene. Expression of the midbrain and floor plate marker genes was confirmed using either miRNA switch (Fig. 5). However, there was no difference in Nurr1 expression between the positive group and the negative group in cells sorted by the miR-218-5p switch (Fig. 5). On the other hand, the expression of Anterior and early nervous system transcription factor was low in the positive group regardless of which miRNA switch was used (Fig. 5). From these results, it was suggested that dopaminergic neural progenitor cells that strongly express midbrain and floor plate marker genes can be FACS-sorted by using either miR-9-5p switch or miR-218-5p switch.
Similarly, on day 21 and day 28 after the initiation of differentiation induction, miR-9-5p switch or miR-218-5p switch was transfected to change the marker expressed by the time from the initiation of differentiation induction. Examined. As a result, the expression pattern of the marker gene was observed using both miR-9-5p switch and miR-218-5p switch, even when the miRNA switch was transfected on the 21st and 28th days from the initiation of differentiation induction. Were similar (Figures 6 and 7).

Comparative Example 1: FACS selection of dopamine neural progenitor cells using anti-CORIN antibody As a comparative example, dopamine neural progenitor cells differentiated from human iPS cells were FACS selected on the 21st and 28th days using anti-CORIN antibody. Then, the expression pattern of the marker gene was examined. Regarding the midbrain and floor plate marker genes, although the expression of CORIN and En1 was confirmed in the cells selected on the 21st day from the initiation of differentiation induction, only the expression of CORIN could be confirmed in the cells selected on the 28th day (Fig. 8). Comparing with the selection result of cells by FACS using miRNA switch, cells that strongly express midbrain and floor plate marker gene were selected by using miRNA switch, and when selecting by the above number of days, It was confirmed that the method of the present invention using the miRNA switch is superior to the method using the anti-CORIN antibody.

Example 4: Expression analysis of marker protein by immunostaining As described above, the marker protein expressed in dopaminergic neural progenitor cells FACS-sorted with the miR-9-5p switch was immunostained and miR-9-5p positive/negative. Comparisons were made between cells and unsorted cells. Compared to miR-9-5p-negative cells and unsorted cells, miR-9-5p-positive cells express Nurr1 and Foxa2, which are marker proteins characteristic of mature dopamine neurons, and are markers of forebrain neural progenitor cells. Expression of the proteins Sox1 and Pax6 was weak, and no difference was observed in the expression of the cell proliferation marker protein Ki67 (Fig. 9).

Example 5: FACS sorting of dopamine neural progenitor cells by tandem miRNA switch From the results obtained so far, it was revealed that the dopamine neural progenitor cells can be sorted with high purity by the sorting using miRNA switch. However, the use of a cell sorter is essential in this approach. Sorting using a cell sorter has drawbacks such as long collection time and low collection efficiency, which makes it unsuitable for sorting a large number of cultured cells. Therefore, the inventors studied a method of selecting cells without using a cell sorter so that the manufactured cells can be used for regeneration treatment in the future.
The gate can be set in the selection using the cell sorter, and even when the transfection efficiency differs between cells, it is possible to perform the sharp selection by using the control EGFP. However, if the cell sorter is not used, generally the gate cannot be set and the transfection efficiency cannot be reflected in the selection. Therefore, it was considered desirable to establish a miRNA switch capable of clearly selecting positive and negative depending on the difference in miRNA expression level. Therefore, the present inventors created tandem miRNA switches in which multiple miRNA target sequences were linked in tandem, and investigated the sensitivity of those tandem miRNA switches.The tandem miR-9-5p switch (1x9-5p-tagBFP-4x9-5p ) Was completed. It was suggested that dopaminergic neural progenitor cells could be FACS-sorted with higher purity when the tandem miR-9-5p switch was used, as compared with the miR-9-5p switch (Fig. 10).

Selection of dopaminergic neural progenitor cells by Bim switch In cells expressing miR-9-5p by linking the gene for apoptosis-inducing protein Bim instead of the fluorescent protein BFP as a tandem miR-9-5p switch marker protein, Bim We attempted to establish a system in which the miRNA switch (Bim switch) that suppresses protein expression functions and is selected by not inducing apoptosis. Here, in order to eliminate cells that are difficult to transfect, a puromycin resistance gene was introduced together with the Bim switch, and selection by puromycin resistance was also performed at the same time.
First, when the optimum amount of puromycin to be added to the medium was examined, it was 5-15 μg/ml, and thereafter, the experiment was conducted at 10 μg/ml (FIG. 12a). Next, the concentration of the puromycin resistance gene required for survival of the cells when 10 μg/ml of puromycin was added to the medium was examined. As a result, it was decided to introduce 100 ng/ml of puromycin resistant mRNA (Fig. 12b). Finally, in a medium supplemented with 10 μg/ml puromycin, cells were severely ligated when various concentrations of Bim switch were introduced together with 100 ng/ml of puromycin resistant mRNA, and when 100 ng/ml of Bim switch was introduced. It was suggested that they could be selected (Fig. 12c).
On the other hand, when the cell recovery rate was examined, it was shown that the method using the Bim switch had a cell recovery rate of about 40%, which was significantly higher than the recovery rate of the FACS-sorted cells (about 10%). (Figure 13b).

Example 6: Comparison of cell recovery efficiency in each method Since cell sorting (purification) is generally performed by FACS sorting of fluorescently labeled cell stains, cell recovery rate by the conventional FACS sorting method is shown. And the cell recovery rates by the selection method of the present invention were compared. When FACS was used to perform FACS selection using miR-9-5p switch and tandem miR-9-5p switch, the detection rates of cells were 14.9% and 28.0%, respectively (Fig. 11). However, this value is the value at the detection part of the FACS machine, and it is said that the cells are actually reseeded and transfected, and then the cells are collected and subjected to the FACS machine to collect further sorted cells. Due to cell loss at each step, not all 14.9% or 28.0% can be recovered at the limit of FACS machine performance. Therefore, based on the number of cells at the time of reseeding, how many target cells could be finally recovered was investigated, and calculated as “collection efficiency”. The cells were reduced to 81% only by unsorting the cells by simply reseeding and collecting the cells the next day without sorting using miRNA switches. The FACS-based method increased the recovery from 6% to 11% by using the tandem miR-9-5p switch than by using the miR-9-5p switch. On the other hand, the recovery rate was 36% in the Bim selection using a drug without cell loss due to the mechanical performance of FACS, and it was shown that the recovery rate was significantly higher by using the Bim switch than when using FACS. ..

Example 7: Expression analysis of various marker proteins/genes in cells selected by Bim switch The expression levels of various marker genes in cells selected by Bim switch as described above were examined. Cells that strongly expressed the midbrain and floor plate marker genes could be selected to the same extent as in the method using FACS described above (Figs. 5 and 14).
In addition, when the expression of the cell marker protein was compared by immunostaining between Bim switch-selected cells and unselected cells, Nur1 which is a marker protein characteristic of mature dopaminergic neurons was found in Bim switch-selected cells. The positive rate was high, and the expression rate of Sox1, a neural progenitor cell marker protein in the forebrain, was low (Fig. 15).

From the above results, it was shown that by using the miRNA switch, cells having a desired phenotype (marker expression type) can be selected from the cell population differentiated from pluripotent stem cells and the like. Furthermore, by using this method, desired cells can be selected without using a cell sorter, which is extremely useful.

Example 8: FACS sorting of cortical neural progenitor cells using miRNA switch
We verified whether this method using miRNA switch can actually be applied to nerve cells other than dopamine neural progenitor cells.
As a result of FACS selection of neural progenitor cells differentiated from mouse ES cells using the miR-9-5p switch, a good selection pattern was confirmed (Fig. 16a). Next, the expression levels of various marker genes in miR-9-5p positive cells were examined. As a result, in miR-9-5p-positive cells, the expression levels of cerebral cortical neural progenitor marker genes Pax6, Ctip2, Emx1 and Fezf2 were high (Fig. 16b), and the expression of mature neural marker genes HES1 and Notch2 was high. The amount was low (Fig. 16c). The expression levels of Sox9, FoxF1 and Sox10, which are other marker genes, were comparable to miR-9-5p-positive cells and unsorted cells, but compared to miR-9-5p-negative cells. It was significantly less (Fig. 16d). From these results, it was suggested that miR-9-5p positive cells were desired cerebral cortical neural progenitor cells and could be well sorted, as in the case of dopamine neural progenitor cells.

From the above results, it is considered that the selection method of the present invention can be applied to various neural progenitor cells other than dopamine neural progenitor cells and cortical neural progenitor cells.

Example 9: Search for miRNA that can be used for selection of neural progenitor cells Differentiation is induced from human iPS cells, and dopamine neural progenitor cells 13 days after initiation are subjected to FACS selection using an anti-CORIN antibody to detect CORIN-positive and negative cells. Was recovered. 642 kinds of miRNA switches (including SEQ ID NOS: 1 to 642) were introduced into these cells, and the responsiveness of each miRNA was analyzed using FACS. Table 8 shows the miRNA sequences used and the results. In the table, DA indicates dopaminergic neural progenitor cells, CN indicates cerebral cortical neural progenitor cells, aa indicates a particularly strong response, a indicates a strong response, b indicates a weak response, and no description indicates no response. Also, the hsa- at the beginning of the miRNA name is omitted.

In dopaminergic neural progenitor cells, among 642 RNA sequences, 16 showed strong response, 36 showed strong response, 76 showed weak response, and 76 showed no response. There were 514 types. Table 9 shows those showing a particularly strong response and those showing a strong response.

In cerebral cortical neural progenitor cells obtained from mouse fetuses, the responsiveness of each miRNA was analyzed using FACS in the same manner as above. In cerebral cortical neural progenitor cells, among 642 types of miRNAs, 14 types showed a particularly strong response, 119 types showed a strong response, 94 types showed a weak response, and no response. There were 415 types. Table 10 shows those showing a particularly strong response and those showing a strong response.

From the above, it was shown that these miRNA switches can also be applied to the present invention.

According to the present invention, donor cells for cell transplantation used for treatment of Parkinson's disease, etc., which can be used as a disease elucidation tool and/or a drug development tool, can be selected using miRNA as an index. It will be possible. In particular, the present invention is excellent in operability, and the labor and time required and the recovery efficiency are significantly improved as compared with the conventional method for selecting neural progenitor cells. Moreover, since the neural progenitor cells produced by the method of the present invention do not use an antibody for selection, there is no risk of residual antibody contamination and scale-up is easy.

(Display of related applications)
This application is based on Japanese Patent Application No. 2017-156213 filed in Japan on Aug. 10, 2017, the entire contents of which are incorporated herein by reference.

Claims (20)

A method for selecting neural progenitor cells, comprising the following steps (1) to (2):
(1) the sequence recognized by miRNA expressed in neural progenitor cells, mRNA functionally linked with a sequence encoding a marker protein, or the step of introducing a DNA encoding the mRNA into the cell, and
(2) A method comprising a step of selecting cells in which expression of the marker protein is suppressed. The method of claim 1, wherein the neural progenitor cell is a dopamine neural progenitor cell. The method according to claim 1, wherein the neural progenitor cells are cerebral cortical neural progenitor cells. The miRNA is miR-9-5p, miR-218-5p, miR-20a-5p, miR-124-3p, miR-7-5p, miR-135a-5p, let-7a-5p, let-7g-5p , Let-7i-5p, miR-26a-5p, miR-106a-5p, miR-320e, miR-374a-5p, let-7b-5p, let-7f-5p, miR-508-5p, miR-20b -5p, miR-30c-5p, miR-30d-5p, miR-30e-5p, miR-135b-3p, miR-17-5p, miR-330-3p, let-7d-5p, miR-93-5p , MiR-106b-5p, miR-335-5p, miR-98-5p, miR-92b-3p, miR-320c, miR-374b-5p, miR-320d, miR-4532, miR-584-3p, miR -219-5p, miR-21-5p, miR-92a-3p, miR-449a, miR-18a-5p, miR-1908-3p, miR-210-5p, miR-34b-5p, miR-34c-3p , MiR-654-5p, miR-660-5p, miR-675-5p, miR-744-3p, miR-942-5p, miR-96-3p, miR-30a-5p, miR-491-5p and miR The method according to claim 2, which is one or more miRNAs selected from the group consisting of -5001-5p. The miRNA is miR-9-5p, let-7a-5p, let-7g-5p, let-7i-5p, miR-330-3p, let-7b-5p, let-7d-5p, let-7f-5p , MiR-135b-3p, miR-508-5p, miR-654-5p, miR-758-5p, miR-491-5p, miR-5001-5p, miR-191-5p, miR-20a-5p, miR -342-3p, miR-99b-5p, miR-423-3p, miR-324-3p, miR-22-3p, miR-378a-3p, miR-296-5p, miR-99a-5p, miR-500a -3p, miR-345-5p, miR-335-5p, miR-134, miR-212-3p, miR-154-5p, miR-150-5p, miR-148a-3p, miR-382-5p, miR -486-5p, miR-370, miR-373-3p, miR-512-5p, miR-516b-5p, miR-518b, miR-518c-5p, miR-519d, miR-523-3p, miR-526a , MiR-98-5p, miR-140-3p, miR-378b, miR-128, miR-3180-3p, miR-1260a, miR-503-5p, miR-34c-5p, miR-3180, miR-21 -3p, miR-4286, miR-4454, miR-138-5p, miR-1307-3p, miR-652-3p, miR-502-3p, miR-92b-5p, miR-501-3p, miR-1285 -3p, miR-126-3p, miR-877-5p, miR-9-3p, miR-129-1-3p, miR-760, miR-365a-5p, miR-374a-5p, miR-4532, miR -20b-3p, miR-363-5p, miR-339-3p, miR-513a-3p, miR-532-3p, miR-3180-5p, miR-4324, miR-1234-5p, miR-329, miR -362-3p, miR-671-5p, miR-24-2-5p, miR-27a-5p, miR-411-5p, miR-197-5p, miR-222-5p, miR-584-3p, miR -326, miR-133a, miR-17-5p, miR-92a-3p, miR-16-5p, miR-339-5p, miR-3 31-3p, miR-203a, miR-298, miR-134-3p, miR-135a-3p, miR-138-1-3p, miR-144-5p, miR-154-3p, miR-15a-5p, miR-15b-3p, miR-15b-5p, miR-16-1-3p, miR-181a-3p, miR-1910-3p, miR-1910-5p, miR-192-3p, miR-194-3p, miR-194-5p, miR-196a-3p, miR-210-5p, miR-34c-3p, miR-450b-3p, miR-4536-3p, miR-4707-5p, miR-490-3p, miR- 494-3p, miR-510-3p, miR-5196-3p, miR-542-5p, miR-574-3p, miR-589-3p, miR-642a-3p, miR-6503-5p, miR-654- One or more miRNA selected from the group consisting of 3p, miR-660-5p, miR-675-5p, miR-766-3p, miR-874-5p, miR-942-5p and miR-96-3p The method of claim 3, wherein: The miRNA is selected from the group consisting of miR-9-5p, let-7a-5p, let-7g-5p, let-7i-5p, let-7b-5p, let-7f-5p and miR-508-5p. The method according to any one of claims 1 to 5, which is one or more miRNAs. 7. The method of any of claims 1-6, wherein the miRNA comprises miR-9-5p. The mRNA comprises, in the 5′ to 3′ direction, one or more sequences recognized by the miRNA, a sequence encoding a marker protein, and one or more sequences recognized by the miRNA. 7. The method according to any one of to 7. The method according to claim 1, wherein the marker protein is a fluorescent protein or an apoptosis-inducing protein. The method according to claim 9, wherein the apoptosis-inducing protein is Bim. The method according to any one of claims 1 to 10, further comprising the step of introducing a nucleic acid encoding a marker protein different from the marker protein into cells. The method according to claim 11, wherein the different marker proteins are fluorescent proteins or drug resistance proteins. The method according to any one of claims 1 to 12, wherein the neural progenitor cell is a cell induced to be differentiated from a pluripotent stem cell. 14. The method of claim 13, wherein the pluripotent stem cells are of human origin. The method according to claim 13 or 14, wherein the neural progenitor cells are sorted on day 22 to 29 after the initiation of differentiation induction. A method for producing neural progenitor cells from pluripotent stem cells, comprising steps (1) to (3) below:
(1) a step of inducing differentiation of neural progenitor cells from pluripotent stem cells,
(2) mRNA obtained by functionally linking a sequence encoding a marker protein to a sequence recognized by miRNAs expressed in neural progenitor cells, or the mRNA obtained in the cell obtained in the step (1) The step of introducing DNA to
(3) A method for producing neural progenitor cells, which comprises a step of selecting cells in which the expression of the marker protein is suppressed. The method according to claim 16, further comprising the step of introducing a nucleic acid encoding a marker protein different from the marker protein into cells. by a miRNA selected from the group consisting of miR-9-5p, let-7a-5p, let-7g-5p, let-7i-5p, let-7b-5p, let-7f-5p and miR-508-5p A kit for selecting neural progenitor cells, comprising mRNA containing a sequence to be recognized and a sequence encoding a marker protein, or DNA containing the mRNA. 19. The mRNA comprises in the 5'to 3'direction one or more sequences recognized by the miRNA, a sequence encoding a marker protein, and one or more sequences recognized by the miRNA. The kit described in. The kit according to claim 18 or 19, further comprising a nucleic acid containing a sequence encoding a marker protein different from the marker protein.