WO2011007847A1 - Method for induction of differentiation of pluripotent stem cells into nerve cells - Google Patents

Abstract

Disclosed is a method for producing nerve cells from pluripotent stem cells, which is characterized by inhibiting the expression or function of Hes1 in the pluripotent stem cell. The method comprises the steps of: (a) bringing the pluripotent stem cells into contact with a substance capable of inhibiting the expression or function of Hes1; and (b) culturing the cells under conditions where the differentiation of the cells into nerve cells can be induced. Also disclosed are: a differentiation inducer comprising a substance capable of inhibiting the expression or function of Hes1; a nerve cell derived from a pluripotent stem cell, which is produced by the method; and a therapeutic agent for nerve diseases, which comprises the nerve cell.

Description

Method for inducing differentiation of neural cells from pluripotent stem cells

The present invention relates to a novel differentiation induction method for producing uniform nerve cells from pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).

The central nervous system neurons of mammals such as mice and humans have poor regenerative ability and are difficult to recover spontaneously once damaged. Therefore, conventional treatments for neurodegenerative diseases and nerve damage such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease have focused on how to reduce neuronal cell death. However, with the development of regenerative medicine, the fundamental treatment method of creating neurons in in vitro culture, replenishing damaged sites and reconstructing neural circuits has become a reality. Therefore, in the field of regenerative medicine, a method for producing nerve cells from pluripotent stem cells such as embryonic stem cells (hereinafter referred to as “ES cells”) and induced pluripotent stem cells (hereinafter referred to as “iPS cells”). Research is ongoing.

Conventionally, differentiation from ES cells to neurons has been performed mainly by the formation of embryoid bodies (EBs) in the presence of serum and selecting EBs containing cells that have differentiated into neurons. Was very low (Non-patent Document 1). In recent years, methods have been developed to increase neuronal differentiation efficiency under co-culture with stromal cells PA6 (Non-patent Document 2) or culture conditions with human amniotic membrane (Non-patent Document 3). Because materials are used, contamination and ethical problems cannot be avoided.
Therefore, a differentiation induction method using a serum-free medium (N2B27 or KSR) having a known chemical composition has been established (Non-Patent Documents 4 and 5). In particular, the method of Ying et al. (Non-Patent Document 4) is superior in that it performs adhesion culture using a medium having a chemical composition, and is relatively unaffected by changes in the culture environment, so that a relatively stable result can be obtained. However, even with this method, (1) it takes more than a week to induce differentiation, (2) the timing of differentiation is uneven, and 20-40% of cells remain undifferentiated without differentiation into the nervous system And the problem of being differentiated in other directions remains. There have been reports of improved methods based on the method of Ying et al., But none have achieved uniform neurons. For example, it has been reported that manipulation to always activate the Notch signal promotes more uniform differentiation into neural progenitor cells (Non-patent Document 6). The terminal differentiation pathway has been inhibited. Although there is a report that the Hedgehog signal contributes to the neuronal differentiation process (Non-patent Document 7), even if the Hedgehog signal is lost, only the survival and proliferation of the cells are reduced, and the differentiation efficiency is not changed. Recently, it has been reported that when the transcription factor REST (NRSF) is knocked down, the neuronal differentiation of a neuronal marker, tuj-1 positive, is increased about twice (Non-patent Document 8). Not touched.

The present invention is to provide a novel differentiation induction method for producing uniform neurons from pluripotent stem cells such as ES cells or iPS cells.

As a result of intensive studies to solve the above problems, the inventors of the present application, as a result of suppressing the gene expression level of Hes1, which is a transcriptional repressor in the embryogenesis stage, in ES cells, has a serum-free composition. The adhesion culture method using a medium succeeded in obtaining nerve cells uniformly within a short period of one week or less, and the present invention was completed.

That is, the present invention is as follows.
[1] A method for producing nerve cells from pluripotent stem cells, which comprises inhibiting the expression or function of Hes1 in pluripotent stem cells.
[2] The method according to [1] above, comprising the following steps.
(A) contacting a pluripotent stem cell with a substance that inhibits the expression or function of Hes1 (b) culturing the cell under conditions that induce differentiation into neuronal cells [3] expression or function of Hes1 The method according to [2] above, wherein the inhibiting substance is any of the following (a) to (h).
(A) antisense nucleic acid against nucleic acid encoding Hes1 (b) siRNA or miRNA for RNA encoding Hes1 or precursor thereof (c) ribozyme nucleic acid for RNA encoding Hes1 (d) Hes1 inhibitor or encoding it Nucleic acid (e) Hes1 decoy nucleic acid (f) Dominant negative mutant of Hes1 or nucleic acid encoding it (g) Antibody against Hes1 or nucleic acid encoding it (h) Nucleic acid that knocks out Hes1 gene by homologous recombination [4] The method according to [2] or [3] above, wherein the step (b) is performed under one or more conditions selected from the following (a) to (c):
(A) Does not induce embryoid body formation (b) Uses a limited medium (c) Does not use a feeder [5] Any of the above [1] to [4], wherein the pluripotent stem cells are ES cells or iPS cells The method of crab.
[6] The method according to any one of [1] to [5] above, wherein the pluripotent stem cells are derived from human or mouse.
[7] A pluripotent stem cell into which a nucleic acid factor that inhibits the expression or function of Hes1 is stably introduced.
[8] A neuron derived from the pluripotent stem cell according to [7] above.
[9] An agent for inducing differentiation of pluripotent stem cells into neurons, comprising any of the following substances (a) to (h):
(A) antisense nucleic acid against nucleic acid encoding Hes1 (b) siRNA or miRNA for RNA encoding Hes1 or precursor thereof (c) ribozyme nucleic acid for RNA encoding Hes1 (d) Hes1 inhibitor or encoding it Nucleic acid (e) Hes1 decoy nucleic acid (f) Dominant negative mutant of Hes1 or nucleic acid encoding it (g) Antibody to Hes1 or nucleic acid encoding it (h) Nucleic acid that knocks out the Hes1 gene by homologous recombination [10] RNA encoding Hes1, which targets a partial nucleotide sequence represented by nucleotide numbers 920 to 940 or 1229 to 1249 in the nucleotide sequence represented by SEQ ID NO: 1, or a sequence corresponding to the partial nucleotide sequence in a Hes1 gene other than mouse SiRNA or precursor thereof.

According to the present invention, nerve cells can be uniformly obtained with very high efficiency as compared with a conventional culture method using a chemical composition medium. In addition, since biological materials with unknown components are not used and formation of complex EBs is not required, differentiation induction can be easily performed on a dish, and mass culture is also possible.

(A) Western blot showing the expression of Hes1 protein in NIH3T3, mouse embryonic fibroblasts (MEF), and ES cells. (B) Western blot (upper) showing expression of Hes1 protein under various culture conditions and quantitative PCR (lower) showing mRNA expression level. Normal ES medium (lane 1), LIFES medium (lane 2), serum-free and LIF-free chemical composition N2B27 medium (lane 3), LIF-containing chemical composition N2B27 medium (lane 4), BMP-containing chemistry The results are shown in composition N2B27 medium (lane 5), chemical composition N2B27 medium (lane 6) containing LIF and BMP, and normal ES medium (lane 7) in the presence of the γ-secretase inhibitor DAPT ((10 μM). (C) Schematic diagram showing regulation of Hes1 expression. (A) shows immunostaining with anti-Hes1 antibody and anti-Oct3 / 4 antibody in mouse ES cells. Nuclei were stained with DAPI. (B) Firefly luciferase under the control of the Hes1 promoter is introduced into a mouse ES cell as a reporter gene to visualize Hes1 expression, and the amount of Hes1 expression in the cell is determined for three mouse ES cells (AC). It is a figure which shows the result of having observed the time-dependent change. (C) It is the figure which measured the luminescence intensity of the luciferase in each ES cell of (B), and plotted with time. It is a figure which shows the half life of Hes1 protein (A) and mRNA (B). (C) Expression of Hes1 protein and mRNA, and changes over time in Hes1 reporter activity. It is a figure which shows a mode that the oscillation of Hes1 expression in ES cell is inherited by two daughter ES cells by division. (A) It is a figure which shows the time-dependent change of Hes1 expression in 5 ES cells, and a power spectrum. (B) It is a figure which shows the cycle of Hes1 expression in each ES cell. (A) Schematic diagram of the targeting construct used for the production of Hes1 knockout cells. (B) A schematic diagram showing the insertion of a loxP sequence for removing exons 2 to 4 of Hes1. (C) and (D) Southern blot analysis to detect homologous recombinants. (E) Schematic diagram showing excision of the second to fourth exons of Hes1 using the Cre-loxP system. (F) Western blot analysis confirming Hes1 inactivation in Hes1 knockout cells. (A) Schematic diagram of the targeting construct used for the production of Hes1 constitutively expressing cells. (B) Southern blot analysis confirming transgene integration. (C) Schematic diagram showing the Neo gene excision using the Cre-loxP system. (A) It is a figure which shows the immunostaining in Hes1 constitutive expression cell (ROSA) using the anti-Hes1 antibody. The fluorescence intensity was visualized in pseudo color (middle panel) and plotted on a histogram (right panel). WT indicates wild type ES cells. (B) Constitutive expression of Hes1 in Hes1 constitutive expression cells (R5 and R6 cells) (lower panel). WT (+ / +) indicates wild type ES cells (upper panel). (C) It is the figure which quantified the result of (B) using LAS-3000 mini (Fuji Film). Each value was standardized with time = 0 as 1. (A) Western blot analysis with anti-Hes1 antibody in Hes1 knockout cells (lanes 2 and 3), Hes1 constitutively expressing cells (lanes 5 and 6), and Hes1 knockdown cells (lanes 8 and 9). . (B) It is the figure which put together the information regarding 7 genes identified as a downstream (target) gene of Hes1 by the microarray analysis using Hes1 knockout cell and Hes1 constitutive expression cell. (C) It is the figure which put together the information regarding the high-order of the Hes1 binding region identified by ChIP-chip analysis. chr is the chromosome number, start and end, and length is the position and length of the binding region. (A) ES cell-specific markers (Oct4, Sox2) in wild-type ES cells (+ / +, +/-), Hes1 knockout cells (K9 / K57 cells), and Hes1 constitutively expressing cells (R5 / R6 cells) Nanog, Rex1, Dppa3, and Dppa4), Hes gene group (Hes3, Hes6, Hey1, and Hey2) and Id gene group (Id1, Id2, and Id3) mRNA expression levels (for wild type (+ / +)) It is a figure which shows (relative amount). (B) mRNA expression level of Hes1 target gene in wild-type ES cells (+ / +, +/-), Hes1 knockout cells (K3 / K9 cells), and Hes1 constitutive expression cells (R5 / R6 cells) It is a figure which shows the relative amount with respect to a wild type. (A) Firefly luciferase under the control of the Hes1 target gene (Dll1, Gadd45g) promoter is introduced into a mouse ES cell as a reporter gene, the expression of each gene is visualized, and the luminescence intensity of luciferase in the cell is measured over time. FIG. (B) Single cell quantitative PCR was performed on 32 ES cells, and the number of copies of expression of Hes1, Dll1, Gadd45g gene, etc. in each cell was plotted. (C) It is the graph which compared the expression of Dll1, Gadd45g gene, etc. for every group at the time of dividing the ES cell of (B) into 3 groups according to Hes1 copy number. (A) Ectodermal / neural markers in wild-type ES cells, K9 / K57 cells, and R5 / R6 cells after differentiation induction under three germ layer differentiation conditions (LIF (-), feeder cells (-)) It is a figure which shows mRNA expression of Mash1, mesoderm marker brachyury, and endoderm marker Gata4. (B) ES cell marker Oct4, ectoderm / neural marker Mash1, neural progenitor cell marker Nestin and neuronal cells in wild-type ES cells, K9 / K57 cells, and R5 / R6 cells after differentiation induction under neural differentiation conditions It is a figure which shows mRNA expression of marker Tuj-1. (C) Immunostaining of Hes1 constitutively expressing cells (R6) induced to differentiate under neural differentiation conditions, and the expression of Oct4 and Nestin after 4 days and the expression of Nestin and Tuj-1 after 6 days are shown. is there. (D) It is a figure which shows the immunostaining which examined the expression of Nestin and Tuj-1 6 days after differentiation induction of the Hes1 knockout cell (K9) on nerve differentiation conditions. (E) Wild type (+/−) and Hes1 knockout cells (K9) are induced to differentiate under neuronal differentiation conditions and show the ratio of Nestin positive and Tuj-1 positive cells 6 days later. The results are shown as an average value ± standard error of 5 fields of confocal images. It is a figure which shows the expression dynamics of Fgf5, brachyury, and Lef1 at the time of nerve differentiation induction in a wild-type ES cell, K9 / K57 cell, and R5 / R6 cell. (A) It is a figure which shows mRNA expression of Hes1 target gene and Notch signal target gene Hes5 at the time of nerve differentiation induction in a wild type ES cell, K9 / K57 cell, and R5 / R6 cell. (B) At the time of neural differentiation induction, the expression of NICD and p57 is up-regulated in Hes1 knockout cells, and the gene expression is down-regulated in Hes1 constitutive expression cells. (C) When Hes1 high-expressing cells (Notch signal off) are delayed in differentiation and differentiation into mesoderm is promoted, while Hes1 low-expressing cells (Notch signal on) are promoted to differentiate into the nervous system. It is a schematic diagram which shows the hypothesis. (A) It is a figure which shows the knockdown efficiency of Hes1 in the human iPS cell (KD1, KD2) which introduce | transduced shRNA with respect to Hes1. As human iPS cells, IMR90-1 and 253G1 strains were used. “Control” indicates human iPS cells into which non-specific shRNA has been introduced. The results are shown as% expression level (average value ± standard error) of Hes1 knockdown cells when the Hes expression level in control is 100. (B) and (C) After induction of differentiation in Hes1 knockdown human iPS cells (KD1, KD2) in IMR90-1 strain (B) or 253G1 strain (C) and control cells (control) into which non-specific shRNA has been introduced It is a figure which shows the mRNA expression of the Sox1 and Tuj-1 gene (B) of the 14th day, or the Sox1 gene (C) of the 7th, 10th, and 14th day of differentiation. “+ Noggin” indicates the results when differentiation is induced in a Noggin-containing medium, and “-Noggin” indicates that the differentiation is induced in a Noggin-free medium. The results are shown as a relative expression level (average value ± standard error) with the expression level in control cells at the start of differentiation induction (day 0) as 1.

The present invention provides a method for producing nerve cells that are more uniform than conventional cells from pluripotent stem cells. Originally, the differentiation mode of pluripotent stem cells such as ES cells is not uniform and is known to progress at various timings and has been considered difficult to control. The mechanism for controlling this variation has not yet been clarified. The present inventors have (i) the expression level of Hes1, which is a transcriptional repressor of basic-helix-loop-helix type, in ES cells periodically oscillates in the cells, and the oscillations are synchronized between the cells. (Ii) The downstream genes that are transcriptionally regulated by Hes1 contain differentiation-promoting transcription factors and Notch signal ligands, and (iii) these downstream genes are also expressed in ES cells. I found that it was vibrating inside. Based on these findings, it was conceived that by controlling and synchronizing the expression or function of Hes1, the variation between ES cells could be controlled, and the expression of Hes1 gene was inhibited in ES cells, and from that cell to the neuron We have succeeded in inducing differentiation of neurons into neurons more uniformly and in a short period of time, and have come to embody this idea. Therefore, the method for producing a nerve cell of the present invention is characterized by inhibiting the expression or function of Hes1 in pluripotent stem cells.

More specifically, the method of the present invention includes the following steps (a) and (b).
(A) A step of contacting a pluripotent stem cell with a substance that inhibits the expression or function of Hes1 (b) A step of culturing the cell under conditions for inducing differentiation into a nerve cell.

(A) A step of contacting a pluripotent stem cell with a substance that inhibits the expression or function of Hes1
(1) Preparation of pluripotent stem cells The pluripotent stem cells used as starting materials have “self-replicating ability” capable of proliferating while maintaining an undifferentiated state and “differentiating pluripotency” capable of differentiating into all three germ layers. If it is an undifferentiated cell, it will not restrict | limit especially, For example, an ES cell, an iPS cell, an embryonic reproductive (EG) cell, an embryonic cancer (EC) cell etc. are mentioned, Preferably it is an ES cell or an iPS cell. The methods of the invention can be applied in any mammal in which any pluripotent stem cell has been established or is capable of being established, for example, human, mouse, rat, monkey, dog, pig, bovine , Cats, goats, sheep, rabbits, guinea pigs, hamsters, etc., preferably humans, mice, rats, monkeys, dogs, etc., more preferably humans or mice.

Pluripotent stem cells can be obtained by methods known per se. For example, as an ES cell production method, for example, a method of culturing an inner cell mass in a mammalian blastocyst stage (see, for example, Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)) ), A method of culturing early embryos produced by somatic cell nuclear transfer (Wilmut et al., Nature, 385, 810 (1997); Cibelli et al., Science, 280, 1256 (1998); Akira Iriya et al., Protein nucleic acid Enzyme, 44, 892 (1999); Baguisi et al., Nature Biotechnology, 17, 456 (1999); Wakayama et al., Nature, 394, 369 (1998); Wakayama et al., Nature Genetics, 22, 127 ( 1999); Wakayama et al., Proc. Natl. Acad. Sci. USA, 96, 14984 (1999); RideoutIII et al., Nature Genetics, 24, 109 (2000)), etc. . Moreover, ES cells can be obtained from a predetermined institution, and further commercially available products can be purchased. For example, human ES cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Regenerative Medicine, Kyoto University.
In the case of somatic cell nuclear transfer, the type of somatic cell and the source from which the somatic cell is collected are in accordance with the following iPS cells.

iPS cells can be prepared by introducing nuclear reprogramming substances into somatic cells. Somatic cells that can be used as a starting material for producing iPS cells may be any cells other than germ cells derived from mammals (eg, mouse or human). There is no particular limitation on the degree of differentiation of cells, and undifferentiated progenitor cells (including somatic stem cells) and terminally differentiated mature cells can be used as the origin of somatic cells.
There are no particular limitations on the mammalian individual from which somatic cells are collected, but from the standpoint that rejection does not occur when the resulting iPS cells are used for the treatment of non-hereditary neurodegenerative diseases in humans. Preferably, somatic cells are collected from the patient himself or others with the same HLA type. On the other hand, when used for the treatment of hereditary neurodegenerative diseases such as Huntington's disease and some Parkinson's disease, collect somatic cells from others who have normal genes and the same HLA type Is preferred. Also, when not being administered (transplanted) to humans, for example, when iPS cells are used as a source of screening cells for evaluating the patient's drug sensitivity and the presence or absence of side effects, It is desirable to collect somatic cells from others who have the same genetic polymorphism that correlates with side effects.
Examples of nuclear reprogramming substances include combinations of various reprogramming genes reported so far (for example, WO 2007/069666, Nature Biotechnology, 26, 101-106 (2008), Cell, 126, 663-676 (2006 ), Cell, 131, 861-872 (2007), Nat. Cell Biol., 11, 197-203 (2009), Nature, 451, 141-146 (2008), Science, 318, 1917-1920 (2007), Stem Cells, 26, 1998-2005 (2008), Cell Research (2008) 600-603, Nature 454: 646-650 (2008), Cell Stem Cell, 2: 525-528 (2008), WO2008 / 118820, Nat. Cell Biol., 11, 197-203 (2009), Nat. Cell Biol., 11, 197-203 (2009), Science, 324: 797-801 (2009)). In addition, a protein encoded by the above reprogramming gene can be introduced into a somatic cell as a nuclear reprogramming substance (Cell Stem Cell, 4: 381-384 (2009), Cell Stem Cell, doi: 10.1016 / j. stem.2009.05.005 (2009)). In particular, a combination of three factors Oct3 / 4, Sox2 and Klf4 is preferred when the obtained iPS cells are used for therapeutic purposes. On the other hand, when iPS cells are not used for therapeutic purposes (for example, as a research tool for drug discovery screening), in addition to the four factors Oct3 / 4, Sox2, Klf4 and c-Myc, Five factors, Oct3 / 4, Klf4, c-Myc, Sox2 and Lin28, 6 factors with Nanog added thereto, and 7 factors with SV40 Large T added thereto are preferred.
iPS cell colonies can be selected using drug resistance and reporter activity as indices (Cell, 126, 663-676 (2006), Nature, 448, 313-317 (2007)) or by visual observation of morphology (Cell, 131, 861-872 (2007)). Confirmation of iPS cells can be performed using various ES cell-specific gene expression and teratoma formation as indicators.

The pluripotent stem cells obtained as described above can be grown while maintaining an undifferentiated state in a medium known per se. As the basal medium, for example, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham's F12 medium, RPMI 1640 medium , Fischer's medium, and mixed media thereof are not particularly limited as long as they can be used for animal cell culture. The medium may be a serum-containing medium or a serum-free medium, but a serum-free medium is preferred from the viewpoint of ensuring the safety of cell transplantation by eliminating different components. Here, a serum-free medium means a medium that does not contain unconditioned or unpurified serum, and a medium that contains purified blood-derived components or animal tissue-derived components (for example, growth factors) is serum-free. It shall correspond to the culture medium. Examples of such serum-free medium include serum-free medium supplemented with an appropriate amount (for example, 1-20%) of commercially available KNOCKOUT ^™ SR (KSR), serum-free medium supplemented with insulin and transferrin (for example, N2B27 [50% DMEM / F12, 50% Neurobasal medium (Gibco), 25 μg / mL insulin, 100 μg / mL apotransferrin, 6 ng / mL progesterone, 16 μg / mL putrescine, 5.2 ng / mL sodium selenite, 1 × B27 (Gibco), 0.1% BSA, 2 mM glutamine], CHO-S-SFM II (GIBCO BRL), Hybridoma-SFM (GIBCO BRL), eRDF Dry Powdered Media (GIBCOBRL), UltraCULTURE ^TM (BioWhittaker), UltraDOMA ^TM ( BioWhittaker), UltraCHO ^™ (BioWhittaker), UltraMDCK ^™ (BioWhittaker), ITPSG medium (Cytotechnology, 5, S17 (1991)), ITSFn medium (Proc. Natl. Acad. Sci. USA, 77, 457) (1980)), mN3 medium (Mech. Dev. 59, 89 (1996), etc.), etc. If necessary, stem cell differentiation inhibitors (eg, LIF, SCF, BMP, Wnt, extracellular matrix, TGF-β, feeder cells) and other components such as amino acids, pyruvate, 2-mercaptoethanol, Cytokines, growth factors and the like may be contained at appropriate concentrations.

The incubator for culturing pluripotent stem cells is not particularly limited as long as it is for cell culture. For example, flask, tissue culture flask, dish, petri dish, tissue culture dish, multi-dish, microplate, microwell plate , Multiplate, multiwell plate, chamber slide, petri dish, tube, tray, culture bag, roller bottle. The incubator may be non-cell-adhesive or cell-adhesive. A cell-adhesive incubator is one in which the surface of the incubator is coated with a cell-supporting substrate for the purpose of improving adhesion to cells (pluripotent stem cells or feeder cells). Examples of the supporting substrate include collagen, gelatin, matrigel, poly-L-lysine, poly-D-lysine, laminin, and fibronectin.
The culture can be performed, for example, in a CO ₂ incubator under an atmosphere having a CO ₂ concentration of about 1 to about 10%, preferably about 2 to about 5%, at about 30 to about 40 ° C., preferably about 37 ° C. .

(2) Substance that inhibits the expression or function of Hes1 “Hes1” in the present invention is a protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2. In the present specification, proteins and peptides are described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation.
Hes1 protein is a cell of humans and other mammals (eg, mice, rats, monkeys, dogs, pigs, cows, rabbits, sheep, goats, etc.) [eg, neurons, glial cells, hepatocytes, spleen cells, pancreas β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, lung cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells) , B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells Or stromal cells, or precursor cells of these cells, stem cells, cancer cells, etc.] or any tissue in which these cells exist [eg, brain, brain regions (eg, olfactory bulb, amygdala, basal sphere, hippocampus, Thalamus, Lower floor, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle (eg, smooth muscle, skeletal muscle), lung, digestion Tube (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, adipose tissue (eg, white adipose tissue, brown adipose tissue) And the like] may be isolated and purified by a protein separation and purification technique known per se.

“Amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2”
(a) an amino acid sequence having about 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2;
(b) an amino acid sequence in which 1 to 20 amino acids are substituted and / or deleted and / or inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2;
(c) the amino acid sequence of an ortholog in another mammal of mouse Hes1 consisting of the amino acid sequence represented by SEQ ID NO: 2; or
(d) Means the amino acid sequence of mouse Hes1 consisting of the amino acid sequence represented by SEQ ID NO: 2 or the orthologue splice variant, allelic variant or polymorphism of (c) above.
As used herein, “homology” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm uses a sequence of sequences for optimal alignment). The percentage of identical and similar amino acid residues relative to all overlapping amino acid residues in which one or both of the gaps can be considered). "Similar amino acids" means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn) ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).

The homology of the amino acid sequences in this specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; filtering) = OFF). Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. Embedded in the program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 1997 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [The algorithm is incorporated into the GAP program in the GCG software package], Myers and Miller, CABIOS, 4: 11-17 (1988) [The algorithm is part of the CGC sequence alignment software package Embedded in the ALIGN program (version 2.0)], Pearson et al., C Proc. Natl. Acad. Sci. USA, 85: 2444-2448 1988 (1988) [the algorithm is a GCG software package. Embedded in the FASTA program in the cage] and the like, and these can be preferably used as well.

In the above (a), more preferably, the “amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2” means about 90% or more, preferably about the amino acid sequence represented by SEQ ID NO: 2. It is an amino acid sequence having about 95% or more, more preferably about 97% or more, particularly preferably about 98% or more.

“A protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2” comprises the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 and SEQ ID NO: 2 A protein having substantially the same activity as a protein comprising the amino acid sequence represented by
Here, “activity” refers to transcriptional control activity of downstream genes such as Dll1 and Gadd45g. Further, “substantially the same quality” means that the properties are qualitatively the same, for example, physiologically or pharmacologically. Therefore, it is preferable that the transcriptional control activity is equivalent, but quantitative factors such as the degree of activity (eg, about 0.1 to about 10 times, preferably about 0.5 to about 2 times) and the molecular weight of the protein are different. Also good.
Hes1 activity can be measured by, for example, measuring and comparing the amount of transcripts of downstream genes in cells that express Hes1 and cells that do not express, reporter genes (such as luciferase) controlled by a promoter containing the Hes1 binding sequence. It can be carried out by examining the suppression of the expression of or the ability to bind to a nucleic acid containing a Hes1-binding sequence.

Further, as Hes1 in the present invention, as shown in the above (b), for example, (i) 1 to 20, preferably 1 to 10, more preferably 1 to several in the amino acid sequence represented by SEQ ID NO: 2 (5, 4, 3, or 2) amino acid sequence in which amino acids are deleted, (ii) 1 to 20, preferably 1 to 10, more preferably 1 to several in the amino acid sequence represented by SEQ ID NO: 2 An amino acid sequence to which (5, 4, 3 or 2) amino acids have been added; (iii) 1 to 20, preferably 1 to 10, more preferably 1 to a number in the amino acid sequence represented by SEQ ID NO: 2 ( 5, 4, 3 or 2) amino acid sequence in which amino acids are inserted; (iv) 1 to 20, preferably 1 to 10, more preferably 1 to number in the amino acid sequence represented by SEQ ID NO: 2 An amino acid sequence in which (5, 4, 3 or 2) amino acids are replaced with other amino acids, or (v) a combination thereof Such protein containing the amino acid sequence are also included.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the transcriptional control activity is maintained.

Preferred examples of the Hes1 protein include, for example, a mouse protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (RefSeq No. NP_032261), or an ortholog thereof (eg, human (SEQ ID NO: 4; RefSeq No) in other mammals. NP_005515), chimpanzee (RefSeq No. XP_516956), cattle (RefSeq No. NP_001029850), rat (RefSeq No. NP_077336), etc., and their splice variants, allele variants, polymorphisms (eg refSNP No. rs11541816 (Leu at position 61 is replaced with Trp)).

In the present invention, the “substance inhibiting Hes1 expression” acts at any stage such as the level of Hes1 gene, the transcription level of Hes1 gene, the level of post-transcriptional regulation, the level of translation into protein, the level of post-translational modification, etc. It may be a thing. Therefore, substances that inhibit Hes1 expression include, for example, substances that deficient in the Hes1 gene, substances that inhibit the transcription of the Hes1 gene (eg, antigenes, hairpin polyamides), and processing from the initial transcript to mRNA. Substances, substances that inhibit the transport of mRNA into the cytoplasm, substances that inhibit the translation of mRNA into protein (eg, antisense nucleic acids, miRNA) or substances that degrade mRNA (eg, siRNA, ribozyme, miRNA), initial translation Examples include substances that inhibit post-translational modifications of the product. Any substance that acts at any stage can be preferably used, but a substance that complementarily binds to mRNA and inhibits translation into a protein or decomposes mRNA is preferable. When cells are used as research tools, it is also preferable to use a substance that deletes the Hes1 gene.

As a substance that specifically inhibits the translation of Hes1 mRNA to protein (or degrades mRNA), preferably contains a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of Hes1 mRNA or a part thereof. A nucleic acid is mentioned.
A nucleotide sequence substantially complementary to the nucleotide sequence of Hes1 mRNA can bind to the target sequence of mRNA and inhibit its translation under physiological conditions of pluripotent stem cells (or cleave the target sequence). Specifically, for example, a region that overlaps with a nucleotide sequence that is completely complementary to the nucleotide sequence of the mRNA (that is, the nucleotide sequence of the complementary strand of the mRNA). About 90% or more, preferably about 95% or more, more preferably about 97% or more, particularly preferably about 98% or more of a nucleotide sequence.
The “nucleotide sequence homology” in the present invention uses the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expectation value = 10; allow gaps; filtering = ON; It can be calculated by match score = 1; mismatch score = -3).

More specifically, the nucleotide sequence complementary or substantially complementary to the nucleotide sequence of Hes1 mRNA is (a) a nucleotide sequence complementary or substantially complementary to the nucleotide sequence represented by SEQ ID NO: 1. Or (b) “a nucleotide sequence that hybridizes under stringent conditions with a complementary strand of the nucleotide sequence represented by SEQ ID NO: 1, substantially comprising a protein comprising the amino acid sequence represented by SEQ ID NO: 2 Examples thereof include a nucleotide sequence that is complementary or substantially complementary to a sequence encoding a protein having the same quality of activity. Here, “substantially the same quality of activity” has the same meaning as described above.
The stringent conditions are, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, such as 6 × SSC (sodium chloride / sodium citrate) / 45 ° C. Hybridization, followed by one or more washes at 0.2 × SSC / 0.1% SDS / 50 to 65 ° C., but those skilled in the art will know the conditions for hybridization that give the same stringency. It can be selected appropriately.

Preferred examples of Hes1 mRNA include mouse Hes1 (RefSeq Accession No. NM_008235) containing the nucleotide sequence represented by SEQ ID NO: 1, or their orthologs in other mammals (eg, human (SEQ ID NO: 3; RefSeq No) NM_005524), chimpanzee (RefSeq No. XM_516956), cattle (RefSeq No. NM_001034678), rat (RefSeq No. NM_024360), etc.), as well as their splice variants, allele variants, polymorphisms (eg refSNP Nos. Rs11541816 , Rs11541817, rs35578304, rs7629144, rs17853878, etc.).

“A part of a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of Hes1 mRNA” means that it can specifically bind to Hes1 mRNA and inhibits the translation of a protein from the mRNA (or the As long as it can degrade mRNA, its length and position are not particularly limited, but from the viewpoint of sequence specificity, at least 10 bases, preferably at least a portion complementary to or substantially complementary to the target sequence, preferably It contains about 15 bases or more, more preferably about 20 bases or more.

Specifically, preferred examples of the nucleic acid comprising a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of Hes1 mRNA, or a part thereof, include any of the following (a) to (c): .
(a) Antisense nucleic acid against Hes1 mRNA
(b) siRNA or miRNA for Hes1 mRNA or a precursor thereof
(c) Ribozyme nucleic acid against Hes1 mRNA

(a) Antisense nucleic acid against Hes1 mRNA The “antisense nucleic acid against Hes1 mRNA” in the present invention is a nucleic acid comprising a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the mRNA, or a part thereof. It has a function of suppressing protein synthesis by forming a specific and stable double strand with the target mRNA and binding.
Antisense nucleic acids are polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages (provided that the polymer is a base such as found in DNA or RNA) And a nucleotide having a configuration that allows attachment of a base). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine etc. ), Sugars (eg, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, metals, radioactive metals) , Boron, an oxidizing metal, etc.), an alkylating agent, and a modified bond (for example, α-anomeric nucleic acid). Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.

As described above, the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA: DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of Hes1. An intron sequence can be determined by comparing a genomic sequence with a Hes1 cDNA nucleotide sequence (eg, SEQ ID NOs: 1 and 3) using a homology search program such as BLAST or FASTA.

The target region of the antisense nucleic acid is not particularly limited in length as long as the antisense nucleic acid hybridizes, and as a result, the translation into the protein is inhibited. The entire sequence of mRNA encoding Hes1 The short sequence may be about 10 bases, and the long sequence may be the entire mRNA or initial transcription product sequence. In view of easiness of synthesis, antigenicity, intracellular migration, etc., an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferred, but is not limited thereto. Specifically, Hes1 5 ′ end hairpin loop, 5 ′ end 6 bp repeat, 5 ′ end untranslated region, translation initiation codon, protein coding region, ORF translation stop codon, 3 ′ end untranslated region, 3 ′ end Paris A nucleotide region or a 3 ′ end hairpin loop or the like may be selected as a preferred target region of an antisense nucleic acid, but is not limited thereto.

In addition, antisense nucleic acids not only hybridize with Hes1 mRNA and early transcription products to inhibit translation into proteins, but also bind to the Hes1 gene, a double-stranded DNA, to form a triplex. In addition, it may be one that can inhibit transcription to RNA (antigene).
Another method for inhibiting the transcription of the Hes1 gene is to use pyrrole-imidazole (PI) polyamide. PI polyamide recognizes the pyrrole (Py) / imidazole (Im) pair as CG, Py / Py pair as AT or TA, and Im / Py pair as GC, which makes it base-specific for various arbitrary double-stranded DNAs And the expression of the target gene can be suppressed. Hydroxypyrrole (Hp) can also be used to discriminate between A and T of the AT base pair. In this case, the Py / Hp pair specifically binds to AT and the Hp / Py pair specifically binds to TA. In addition, there is a slight pitch shift between the PI polyamide pair and the base pair. To eliminate this, a flexible β-alanine / β-alanine pair for AT base pairs every 3 or 4 base pairs. Can be introduced. Furthermore, when PI polyamide chains that bind to complementary DNA strands are linked via γ-aminobutyric acid (GABA), the molecule is bent into a hairpin type and the γ-turn portion has strong AT base pair selectivity. Design PI polyamides, preferably hairpin polyamides, that specifically bind to the transcription factor binding region in the Hes1 promoter, as PI polyamides penetrate and bind to DNA minor grooves, thereby inhibiting transcription factor binding. Thus, the expression of Hes1 can be suppressed by inhibiting RNA transcription from the Hes1 gene. As a specific target region in a promoter that inhibits transcription of the Hes1 gene, for example, (1) a transcription start point [base sequence from immediately before the start ATG codon of mouse Hes1 to 500 bases upstream represented by SEQ ID NO: 11 (Base number 1 is -500 position, base number 500 is equivalent to position -1 (the "A" of the starting ATG is +1)), the base represented by base number 254 (or 251)] and TATA- box (in the base sequence represented by SEQ ID NO: 11, the base sequence represented by base numbers 223-229) and its peripheral region, (2) the binding site of RBP-Jκ (in the base sequence represented by SEQ ID NO: 11, Base numbers 166-176 and 191-199) and their peripheral regions, (3) Nanog binding sites (base numbers 74-80 and 151-157 in the base sequence represented by SEQ ID NO: 11) and their peripheral regions, etc. It is done. The sequence of the Hes1 promoter region including the regions (1) to (3) is very well conserved between human and mouse.

The nucleotide molecule constituting the antisense nucleic acid may be natural DNA or RNA, but various chemicals may be used to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA). Modifications can be included. For example, in order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue. In addition, the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R═CH ₃ (2′-O-Me), CH ₂ CH ₂ OCH ₃ (2′-O-MOE), CH ₂ CH ₂ NHC (NH) NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN, etc.) may be substituted. Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned.

The conformation of the sugar part of RNA is dominated by C2'-endo (S type) and C3'-endo (N type). In single-stranded RNA, it exists as an equilibrium between the two, but double-stranded Is fixed to the N type. Therefore, in order to give strong binding ability to the target RNA, BNA (LNA) (Imanishi) is an RNA derivative in which the conformation of the sugar moiety is fixed to N-type by cross-linking 2 'oxygen and 4' carbon. , T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides Nucleotides Nucleicides Nucleicides Nucleicides , 22, 1619-21, 2003) can also be preferably used.

The antisense oligonucleotide of the present invention determines the target sequence of mRNA or initial transcription product based on the cDNA sequence or genomic DNA sequence of Hes1, and commercially available DNA / RNA automatic synthesizers (Applied Biosystems, Beckman, etc.) ) To synthesize a sequence complementary thereto. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a method known per se.

(b) siRNA or miRNA against Hes1 mRNA
In the present specification, double-stranded RNA consisting of oligo RNA complementary to Hes1 mRNA and initial transcript and its complementary strand, so-called siRNA and miRNA, is also complementary or substantially complementary to the nucleotide sequence of Hes1 mRNA. Defined as encompassed by a nucleic acid comprising a complementary nucleotide sequence or a portion thereof. When a short double-stranded RNA is introduced into a cell, the mRNA complementary to the RNA is degraded. So-called RNA interference (RNAi) has long been known in nematodes, insects, plants, etc. Since this phenomenon was confirmed to occur widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative to ribozyme.

siRNA can be designed according to the rule proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)) based on the cDNA sequence or genomic DNA sequence information of Hes1, for example. Examples of the target sequence of siRNA include, but are not limited to, AA + (N) ₁₉ , AA + (N) ₂₁ or NA + (N) ₂₁ (N is an arbitrary base). The position of the target sequence is not particularly limited. For the selected target sequence candidate group, whether or not there is homology in the 16-17 base sequence in the non-target mRNA is determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) And the like, and the specificity of the selected target sequence is confirmed. For example, when AA + (N) ₁₉ , AA + (N) ₂₁ or NA + (N) ₂₁ (N is an arbitrary base) is used as a target sequence, the target sequence whose specificity has been confirmed is AA (or NA) or later. Two strands consisting of a sense strand having a TT or UU 3 'end overhang at 19-21 bases and an antisense strand having a sequence complementary to the 19-21 base and a TT or UU 3' end overhang Strand RNA may be designed as siRNA. In addition, for short hairpin RNA (shRNA) which is a precursor of siRNA, an arbitrary linker sequence (for example, about 5-25 bases) capable of forming a loop structure is appropriately selected, and the sense strand and the antisense strand are combined with each other. It can be designed by linking via a linker sequence.

The sequence of siRNA and / or shRNA can be searched using search software provided free of charge on various websites. Examples of such sites include siRNA Target Finder (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) and pSilencer ^™ Expression Vector insert design tool (http: / /www.ambion.com/techlib/misc/psilencer_converter.html) and GeneSeer (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) provided by RNAi Codex are not limited to these. Moreover, siRNA and shRNA against Hes1 mRNA are commercially available from, for example, Invitrogen.

Examples of particularly preferred siRNAs of the present invention include partial nucleotide sequences represented by nucleotide numbers 920 to 940 (SEQ ID NO: 7) or 1229 to 1249 (SEQ ID NO: 8) in the nucleotide sequence represented by SEQ ID NO: 1, and other than mouse Examples include siRNA targeting a sequence corresponding to the partial nucleotide sequence in the Hes1 gene. For example, in the case of the human Hes1 gene, the target sequence corresponding to the partial nucleotide sequence is a partial nucleotide sequence represented by nucleotide numbers 916 to 936 or 1230 to 1252 in the nucleotide sequence represented by SEQ ID NO: 3. Alternatively, as another preferable siRNA for human Hes1 mRNA, a partial nucleotide sequence represented by nucleotide numbers 1255-1275 (SEQ ID NO: 9) or 327-347 (SEQ ID NO: 10) in the nucleotide sequence represented by SEQ ID NO: 3 is targeted. SiRNA to be used. SiRNAs targeting these partial nucleotide sequences have significantly higher Hes1 knockdown activity than conventionally commercially available siRNAs.

MicroRNA (miRNA) is a natural double-stranded RNA small molecule consisting of 18 to 25 bases that binds complementarily to a specific region of a target mRNA and controls translation into the protein. Examples of miRNA that targets Hes1 mRNA and inhibits its translation into protein include miR-9.

The ribonucleoside molecules constituting siRNA and miRNA may also be modified in the same manner as in the above-described antisense nucleic acid in order to improve stability, specific activity and the like.

For siRNA and miRNA, the sense strand and antisense strand of the target sequence on mRNA were synthesized with a DNA / RNA automatic synthesizer and denatured at about 90 to about 95 ° C for about 1 minute in an appropriate annealing buffer. Thereafter, it can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. It can also be prepared by synthesizing a single hairpin RNA (shRNA) or pre-miRNA that is a precursor of siRNA or miRNA, and cleaving it with a dicer.

As used herein, a nucleic acid designed to generate siRNA or miRNA against Hes1 mRNA in vivo also includes a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of Hes1 mRNA, or a part thereof. It is defined as encompassed by the containing nucleic acid. Examples of such a nucleic acid include the above-mentioned shRNA, pre-miRNA or pri-miRNA, or an expression vector constructed so as to express siRNA (shRNA) or miRNA (pre-miRNA, pri-miRNA). shRNA is an oligo comprising a nucleotide sequence in which a sense sequence and an antisense strand of a target sequence on mRNA are inserted by interposing a spacer sequence (for example, about 5 to 25 bases) long enough to form an appropriate loop structure. It can be prepared by designing RNA and synthesizing it with an automatic DNA / RNA synthesizer. Vectors expressing shRNA include tandem type and stem loop (hairpin) type. In the former, siRNA sense strand expression cassette and antisense strand expression cassette are linked in tandem, and each strand is expressed and annealed in the cell to form double stranded siRNA (dsRNA). It is. On the other hand, the latter is one in which an shRNA expression cassette is inserted into a vector, in which shRNA is expressed in cells and processed by dicer to form dsRNA. As the promoter, a pol II promoter (for example, a CMV immediate early promoter) can be used, but in order to accurately transcribe a short RNA, a pol III promoter is generally used. Examples of polIII promoters include mouse and human U6-snRNA promoters, human H1-RNase P RNA promoter, and human valine-tRNA promoter. Further, a sequence in which 4 or more Ts are continuous is used as a transcription termination signal. An expression cassette for miRNA or a precursor thereof can be constructed in the same manner.
The siRNA (shRNA) or miRNA (pre-miRNA, pri-miRNA) expression cassette thus constructed is then inserted into a plasmid vector or viral vector. As such vectors, viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, Sendai viruses, animal cell expression plasmids, episomal vectors capable of autonomous replication in animal cells, and the like are used.

(c) Ribozyme nucleic acid against Hes1 mRNA As another preferred example of a nucleic acid containing a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of Hes1 mRNA or the nucleotide sequence of the initial transcript or a part thereof, the RNA is specific. And ribozyme nucleic acids that can be cleaved. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acids in a narrow sense, but in this specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleavage activity. The most versatile ribozyme nucleic acids include self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known. The hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends (about 10 bases in total) adjacent to the part having the hammerhead structure are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate. When Hes1 mRNA has a double-stranded structure by itself, the target sequence can be made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when ribozymes are used in the form of expression vectors containing the DNA that encodes them, they should be hybrid ribozymes in which tRNA-modified sequences are further linked in order to promote the transfer of transcripts to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

Nucleic acids containing a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of Hes1 mRNA or a part thereof are provided in a special form such as a liposome or microsphere, or in a form to which other molecules are added. Can be provided. Additional forms used include polycationic substances such as polylysine that act to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake (eg , Phospholipids, cholesterol, etc.). Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.). These molecules can be added to the 3 'or 5' end of nucleic acids and can be added via bases, sugars, intramolecular nucleoside linkages. Examples of other modifying groups include capping groups that are specifically arranged at the 3 'end or 5' end of nucleic acids to prevent degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

The substance that inhibits the expression of Hes1 in the present invention is a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of Hes1 mRNA (including an initial transcript or genomic DNA in some cases) as described above or one of them. The substance is not limited to a nucleic acid containing a portion, and may be another substance such as a low molecular weight compound as long as it directly or indirectly inhibits the production of Hes1 protein. Examples of such substances include γ-secretase modulators such as non-steroidal anti-inflammatory drugs (NSAIDs).

In the present specification, substances that delete the Hes1 gene (eg, insertion mutation, deletion mutation) are also included in the “substances that inhibit the expression of Hes1” in the present invention. Examples of the substance that deletes the Hes1 gene include a nucleic acid that knocks out the Hes1 gene by homologous recombination.

In the present invention, the “substance that inhibits the function of Hes1” may be any substance as long as the functionally produced Hes1 protein inhibits the control of transcription of the downstream gene group. For example, it binds to the Hes1 protein. And substances that inhibit the binding to the downstream gene, substances that bind to the downstream gene competitively with the Hes1 protein, and do not control the transcription of the gene.

Specifically, as a substance that inhibits the function of Hes1,
(d) Hes1 inhibitor or nucleic acid encoding it
(e) Hes1 decoy nucleic acid
(f) Dominant negative mutant of Hes1 or nucleic acid encoding it
(g) An antibody against Hes1 or a nucleic acid encoding it.

(d) Hes1 inhibitor or nucleic acid encoding it Hes1 inhibitor includes, for example, Hes6 as a protein factor and QLT0267, LY294002, etc. as non-protein compounds. Hes1 forms a homodimer and binds to a consensus sequence (N-box: CACNAG or E-box: CANNTG) present in the regulatory region of the downstream gene, while Hes6 binds to Hes1 and binds to the downstream gene of Hes1. Inhibit. Hes6 protein can be purified from cells that produce it using known protein separation and purification techniques, or known peptide synthesis based on known amino acid sequence information (for example, Refseq No. NP_061115 of human Hes6). It can be prepared by chemically synthesizing according to the method or by cloning Hes6 cDNA based on known nucleotide sequence information (for example, Refseq No. NP_018645 of human Hes6) to produce a recombinant protein. Recombinant Hes6 protein is commercially available from, for example, Abnova. When the Hes1 inhibitor is a protein factor such as Hes6, a nucleic acid encoding the protein can also be used. For example, in the case of Hes6, the Hes6 cDNA obtained as described above can be used.

(e) Hes1 decoy nucleic acid As described above, Hes1 binds to a consensus sequence (N-box: CACNAG or E-box: CANNTG) present in the regulatory region of the downstream gene. Therefore, oligonucleic acid (eg, double-stranded DNA, double-stranded RNA, DNA / RNA hybrid) containing the consensus sequence can suppress its function by inhibiting the binding of Hes1 to the target gene. . The Hes1 decoy nucleic acid can be prepared by the same method as the siRNA for Hes1.

(f) The dominant negative mutant of Hes1 or the nucleic acid that encodes it The dominant negative mutant of Hes1 (hereinafter referred to as D / N-Hes1) forms a dimer with wild-type Hes1, but the regulatory region of the downstream gene Can not bind to Hes1, so the function of Hes1 can be inhibited. As D / N-Hes1, for example, one or more polar amino acids in the basic region are replaced with non-polar amino acids (eg, Ala, Val, Leu, Ile, etc.) (eg, Glu at position 43, position 44) And a mutant in which Arg at position 47 is substituted with Ala (E43A / K44A / R47A)). D / N-Hes1 can be obtained by the following method, for example. First, an appropriate oligonucleotide is synthesized as a probe or primer based on the mouse or human Hes1 cDNA sequence information shown in SEQ ID NO: 1 or 3, and the mouse, human cell or tissue-derived mRNA, cDNA or cDNA library is used. The mouse or human Hes1 cDNA is cloned using a hybridization method or (RT-) PCR method and subcloned into an appropriate plasmid. The codon of the site to introduce the mutation (for example, in the case of E43A / K44A / R47A, gag aag agg cga agg (SEQ ID NO: 5) represented by nucleotide numbers 361 to 375 in the nucleotide sequence represented by SEQ ID NO: 1) Synthesizing a primer containing the site in a form substituted with a codon encoding a desired other amino acid (for example, in the case of E43A / K44A / R47A, g c g gc g agg cga gc g (SEQ ID NO: 6)); By using this, inverse PCR using a plasmid inserted with Hes1 cDNA as a template, a nucleic acid encoding the target dominant negative mutant is obtained. When D / N-Hes1 is used in the form of protein for Hes1 inhibition, the nucleic acid is inserted into an appropriate expression vector, introduced into a host cell, and the cell is cultured to obtain D / N-Hes1 protein. be able to. Alternatively, a dominant negative mutant can be similarly produced by deleting a basic region essential for DNA binding.

(g) An antibody against Hes1 or another substance that inhibits the function of nucleic acid Hes1 encoding it, for example, an antibody against Hes1. The antibody may be a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to per se known antibody or antiserum production methods. The isotype of the antibody is not particularly limited, but preferably IgG, IgM or IgA, particularly preferably IgG. The antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding a target antigen. In addition to a complete antibody molecule, for example, Fab, Fab ′, F (ab ') ₂ such as fragments, scFv, scFv-Fc, conjugation molecules prepared by genetic engineering such as minibodies and diabodies, or molecules having protein stabilizing action such as polyethylene glycol (PEG) They may be modified derivatives thereof. Considering that Hes1 is a transcriptional repressor and has nuclear localization, it is desirable to use a low molecular weight antibody molecule.

(3) Contact between a pluripotent stem cell and a substance that inhibits the expression or function of Hes1 Contact between a pluripotent stem cell and a substance that inhibits the expression or function of Hes1 (hereinafter sometimes referred to as a “Hes1 inhibitor”) Can be performed using known methods for introducing proteins into cells when the Hes1 inhibitor is a protein factor (eg, a protein Hes1 inhibitor, D / N-Hes1, an antibody to Hes1, etc.) . When the neuronal cells that are induced to differentiate from pluripotent stem cells according to the present invention are intended for human clinical application such as treatment of neurodegenerative diseases, it is preferable that the cells be produced without genetic manipulation.

Examples of such a method include a method using a protein introduction reagent, a method using a protein introduction domain (PTD) or a cell-penetrating peptide (CPP) fusion protein, and a microinjection method. Protein introduction reagents include cationic lipid-based BioPOTER Protein Delivery Reagent (Gene Therapy Systmes), Pro-Ject ^™ Protein Transfection Reagent (PIERCE) and ProVectin (IMGENEX), and lipid-based Profect-1 (Targeting Systems) ), Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif) based on a membrane-permeable peptide, GenomONE (Ishihara Sangyo) using HVJ envelope (inactivated Sendai virus), and the like are commercially available. The introduction can be carried out according to the protocol attached to these reagents, but the general procedure is as follows. Dilute the Hes1 inhibitor in an appropriate solvent (for example, buffer solution such as PBS, HEPES, etc.), add the introduction reagent, incubate at room temperature for about 5-15 minutes to form a complex, and replace it with serum-free medium. And incubate at 37 ° C. for 1 to several hours.

As PTD, Drosophila-derived AntP, HIV-derived TAT (Frankel, A. et al, Cell 55, 1189-93 (1988); Green, M. & Loewenstein, PM Cell 55, 1179-88 (1988)), Penetratin (Derossi, D. et al, J. Biol. Chem. 269, 10444-50 (1994)), Buforin II (Park, CB et al. Proc. Natl Acad. Sci. USA 97, 8245-50 (2000) ), Transportan (Pooga, M. et al. FASEB J. 12, 67-77 (1998)), MAP (model amphipathic peptide) (Oehlke, J. et al. Biochim. Biophys. Acta. 1414, 127-39 ( 1998)), K-FGF (Lin, YZ et al. J. Biol. Chem. 270, 14255-14258 (1995)), Ku70 (Sawada, M. et al. Nature Cell Biol. 5, 352-7 (2003) )), Prion (Lundberg, P. et al. Biochem. Biophys. Res. Commun. 299, 85-90 (2002)), pVEC (Elmquist, A. et al. Exp. Cell Res. 269, 237-44 ( 2001)), Pep-1 (Morris, MC et al. Nature Biotechnol. 19, 1173-6 (2001)), Pep-7 (Gao, C. et al. Bioorg. Med. Chem. 10, 4057-65 ( 2002)), SynBl (Rousselle, C. et al. MoI. Pharmacol. 57, 679-86 (2000)), HN-I (Hong, FD & Clayman, GL. Cancer Res. 60, 6551 -6 (2000)), HSV-derived proteins such as VP22 have been developed using the cell translocation domain. Examples of CPP derived from PTD include polyarginine such as 11R (Cell Stem Cell, 4: 381-384 (2009)) and 9R (Cell Stem Cell, 4: 472-476 (2009)).
A fusion protein expression vector incorporating a Hes1 inhibitor cDNA and a PTD or CPP sequence is prepared and recombinantly expressed, and the fusion protein is recovered and used for introduction. The introduction can be performed in the same manner as described above except that no protein introduction reagent is added.

Microinjection is a method in which a protein solution is put into a glass needle having a tip diameter of about 1 μm and puncture is introduced into a cell, and the protein can be reliably introduced into the cell.

However, if importance is placed on the inhibition efficiency of Hes1 expression or function, the Hes1 inhibitor is preferably used in the form of a nucleic acid that encodes it rather than as a protein factor itself. The nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. The nucleic acid may be double-stranded or single-stranded. Preferably, the nucleic acid is double stranded DNA, in particular cDNA.

DNA encoding a Hes1 inhibitor (eg, protein Hes1 inhibitor, D / N-Hes1, antibody against Hes1, etc.) is inserted into an appropriate expression vector containing a promoter that can function in the host pluripotent stem cell. The Examples of expression vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sendai virus and other viral vectors, animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV). , PcDNAI / Neo) or the like.

The type of vector to be used can be appropriately selected according to the use of the nerve cell obtained by inducing differentiation of the pluripotent stem cell. For example, adenovirus vectors, plasmid vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, Sendai virus vectors, episomal vectors and the like can be used.

Examples of the promoter used in the expression vector include EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Molone murine leukemia virus) LTR. HSV-TK (herpes simplex virus thymidine kinase) promoter and the like are used. Of these, EF1α promoter, CAG promoter, MoMuLV LTR, CMV promoter, SRα promoter and the like are preferable.

In addition to the promoter, the expression vector may contain an enhancer, a poly A addition signal, a selection marker gene, an SV40 replication origin, and the like as desired. Examples of the selection marker gene include a dihydrofolate reductase gene, a neomycin resistance gene, a puromycin resistance gene, and the like.

An expression vector containing a nucleic acid encoding a Hes1 inhibitor can be introduced into cells by a method known per se, depending on the type of vector. For example, in the case of a viral vector, a virus produced in the culture supernatant by introducing a plasmid containing the nucleic acid into an appropriate packaging cell (eg, Plat-E cell) or a complementary cell line (eg, 293 cell) The vector is collected and cells are infected with the vector by an appropriate method according to each viral vector. When using the pluripotent stem cells as a cell source for regenerative medicine, the expression (reactivation) of Hes1 inhibitor may increase the risk of carcinogenesis in neural tissue regenerated from pluripotent stem cell-derived neurons Therefore, it is preferable that the nucleic acid encoding the Hes1 inhibitor is transiently expressed without being integrated into the cell chromosome. From this point of view, it is preferable to use an adenovirus vector that rarely integrates into the chromosome. In addition, adeno-associated virus also has a low frequency of integration into chromosomes, and has lower cytotoxicity and inflammation-inducing action than adenovirus vectors, and thus can be mentioned as another preferred vector. The Sendai virus vector can exist stably outside the chromosome, and can be preferably used in the same manner because it can be decomposed and removed by siRNA as necessary. As the Sendai virus vector, those described in J. Biol. Chem., 282, 27273-27391 (2007) can be used.

When using a retrovirus vector or lentivirus vector, even if silencing of the transgene occurs, it may be reactivated later, so it became unnecessary, for example, using the Cre / loxP system A method of excising a nucleic acid encoding a Hes1 inhibitor at the time can be preferably used. That is, loxP sequences are arranged at both ends of the nucleic acid, and after induction of differentiation into neurons, Cre recombinase is allowed to act on the cells using a plasmid vector or an adenovirus vector, so that the region sandwiched between the loxP sequences Can be cut out. In addition, the enhancer-promoter sequence in the LTR 領域 U3 region may up-regulate nearby host genes by insertion mutation, so the 3′-self is deleted or replaced with a polyadenylation sequence such as SV40. More preferably, an inactivated (SIN) LTR is used to avoid expression control of the endogenous gene by an LTR outside the loxP sequence that is not excised and remains in the genome.

On the other hand, in the case of a plasmid vector which is a non-viral vector, the vector is transferred to cells using lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, DEAE dextran method, microinjection method, gene gun method, etc. Can be introduced.

When a plasmid vector, adenovirus vector, or the like is used, gene transfer can be performed any number of times of 1 or more (for example, 1 to 10 times, or 1 to 5 times).

Even when an adenovirus or plasmid is used, since the transgene may be integrated into the chromosome, use a means for removing the gene once the transgene has been integrated into the chromosome, as in the Cre-loxP system. It can be convenient. In another preferred embodiment, there is a method for completely removing a transgene from a chromosome by incorporating a transgene into a chromosome using a transposon and then allowing a transferase to act on the cell using a plasmid vector or an adenovirus vector. Can be used. Preferred transposons include, for example, piggyBac, which is a transposon derived from a lepidopteran insect.

Another preferred non-integrated vector is an episomal vector capable of autonomous replication outside the chromosome. Examples of the episomal vector include a plasmid containing a nucleic acid encoding Epstein-Barr virus (EBV) origin of replication oriP and a protein EBNA-1 which binds to the replication origin and controls replication.

Hes1 inhibitor is a short nucleic acid molecule (eg, antisense nucleic acid against Hes1-encoding nucleic acid, siRNA or miRNA or precursor to RNA encoding Hes1, ribozyme nucleic acid against RNA encoding Hes1, Hes1 decoy nucleic acid, etc.) In some cases, as in the case of plasmid DNA, contact between pluripotent stem cells and Hes1 inhibitor is introduced into cells using liposome method, polyamine method, electroporation method, bead method, etc. Can be implemented. The method using cationic liposomes is the most common, and the introduction efficiency is high. In addition to common gene introduction reagents such as Lipofectamine 2000 and Oligofectamine (Invitrogen), introduction reagents suitable for siRNA introduction such as GeneEraser ^™ siRNA transfection reagent (Stratagene) are also commercially available.

When the Hes1 inhibitor is provided in the form of a vector that expresses the short nucleic acid molecule, the contact of the vector with the pluripotent stem cell is a plasmid vector prepared as described in (2) (b) above, This is performed by introducing a viral vector or episomal vector into a cell. These gene introductions can be performed in the same manner as described above for introduction of a nucleic acid encoding a proteinous Hes1 inhibitor.

When the Hes1 inhibitor is a low molecular weight compound, the substance is introduced into somatic cells by dissolving the substance in an aqueous or non-aqueous solvent at an appropriate concentration, and a medium suitable for culturing pluripotent stem cells (described above) In a medium suitable for inducing differentiation into cells and neurons (described later), the Hes1 inhibitor concentration is sufficient to significantly inhibit the expression or function of Hes1 in pluripotent stem cells and is not cytotoxic. Thus, the inhibitor solution can be added, and the cells can be cultured for a certain period of time. The Hes1 inhibitor concentration varies depending on the type of Hes1 inhibitor used, but is appropriately selected within the range of about 0.1 nM to about 100 nM.

When the Hes1 inhibitor is a nucleic acid that knocks out the Hes1 gene by homologous recombination, the specific method for knocking out the Hes1 gene is to isolate the Hes1 gene from the genomic DNA of the animal from which the pluripotent stem cells are derived according to conventional methods. For example, (1) the function of the exon or promoter is destroyed by inserting another DNA fragment (for example, drug resistance gene or reporter gene) into the exon part or promoter region, or (2) Cre-loxP system Or the Flp-frt system to excise all or part of the Hes1 gene and delete the gene, or (3) insert a stop codon into the Hes1 coding region to disable complete translation of the Hes1 protein. Or (4) by inserting a DNA sequence (for example, a polyA addition signal) that terminates gene transcription into the transcription region and disabling complete mRNA synthesis. For example, a method of incorporating a DNA strand (targeting vector) having a DNA sequence constructed so as to inactivate the Hes1 gene into the Hes1 locus of pluripotent stem cells by homologous recombination can be preferably used.

Usually, gene recombination in mammals is mostly non-homologous, and the introduced DNA is randomly inserted at any position on the chromosome. Therefore, depending on selection (positive selection) such as detection of drug resistance and reporter gene expression, it is not possible to efficiently select only clones targeted to the target Hes1 gene by homologous recombination. For these clones, it is necessary to confirm the integration site by Southern hybridization or PCR. Therefore, if a herpes simplex virus-derived thymidine kinase (HSV-tk) gene that confers sensitivity to ganciclovir, for example, is linked outside the region homologous to the target sequence of the targeting vector, cells into which the vector is randomly inserted Has an HSV-tk gene and cannot grow in a ganciclovir-containing medium, but cells targeted to the Hes1 locus by homologous recombination do not have the HSV-tk gene and are selected to be resistant to ganciclovir (negative selection). Alternatively, if, for example, a diphtheria toxin gene is linked instead of the HSV-tk gene, cells into which the vector has been randomly inserted will be killed by the toxin produced by the vector, so that homologous recombinants can be removed in the absence of a drug. You can also choose.

For introduction of targeting vectors into pluripotent stem cells, calcium phosphate coprecipitation method, electroporation method, lipofection method, retrovirus infection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, etc. As described above, gene recombination in mammals is mostly non-homologous and the frequency with which homologous recombinants are obtained is low, so that a large number of cells can be treated easily. The electroporation method is generally selected. For electroporation, the conditions used for gene transfer into normal animal cells may be used as they are.For example, after pluripotent stem cells in the logarithmic growth phase are treated with trypsin and dispersed into single cells, It can be carried out by suspending in a medium so as to be 10 ⁶ to 10 ⁸ cells / ml, transferring to a cuvette, adding 10 to 100 μg of the targeting vector, and applying an electric pulse of 200 to 600 V / cm.

Pluripotent stem cells into which a targeting vector has been incorporated can also be assayed by screening chromosomal DNA isolated and extracted from colonies obtained by culturing single cells by Southern hybridization or PCR.

The pluripotent stem cell obtained by knocking out the Hes1 gene obtained as described above can be used as it is for inducing differentiation into the nerve cell of the present invention, or if the pluripotent stem cell is derived from a non-human mammal. In accordance with a conventional method, a chimeric animal is prepared, a germline chimera is selected, and the resulting germline chimeric animal is crossed as a founder, and a Hes1 knockout homozygote is established and maintained. ES cells may be established from knockout animals according to conventional methods and used for differentiation induction.

(B) Step of inducing differentiation of pluripotent stem cells in which the expression or function of Hes1 is inhibited The pluripotent stem cells in which the expression or function of Hes1 is inhibited (hereinafter referred to as “Hes1 inactivity”) obtained by the above step (a). Can be induced to differentiate into nerve cells by any method. “Nerve cell” means a cell having a function of transmitting a stimulus to another nerve cell, muscle or gland cell upon receiving a stimulus from another nerve cell or a stimulus receptor cell, It is categorized by the difference in secreting neurotransmitters. Neurons classified by these neurotransmitters are classified into, for example, dopamine secreting neurons, acetylcholine secreting neurons, serotonin secreting neurons, noradrenaline secreting neurons, adrenergic secreting neurons, glutamate secreting neurons, and the like. . In the present invention, not only mature nerve cells but also neural progenitor cells determined to differentiate into the nervous system among the ectoderm cells are included in the “nerve cells”. Specifically, “neural cells” in the present invention are defined as Nestin-1 positive cells. Preferably, the nerve cell is further βIII-tubulin (Tuj-1) positive.

As a method for inducing differentiation of neuronal cells from pluripotent stem cells, for example, EB formation method (Okabe S et al. Mech. Dev. 59 89-102 (1996); Lee SH et al. Nature Biotech. 18 675-9 (2000)), retinoic acid method (Bain G et al. Dev. Biol. 168 342-57 (1995)), SDIA (stromal cell-derived inducing activity) method (Kawasaki H et al. Neuron 28 31-40 (2000) )), Serum-free monolayer adherent culture method (Ying QL et al. Nature Biotech. 21 183-186 (2003); Ying QL and Smith AG Methods Enzymol. 365 327-41 (2003)), SFEB (serum-free floating culture of embryonic body-like aggregation) method (Watanabe K et al. Nature Neurosci. 8 288-96 (2005)), AMED (amniotic membrane matrix-based ES cell differentiation) method (Ueno M et al. Proc. Natl. Acad Sci. USA 103 9554-9 (2006)), NSS (neural stem sphere) method (Nakayama T et al. Neurosci. Res. 46 241-9 (2003)), etc. But it is not limited. However, in light of the main purpose of the present invention to induce differentiation into a more uniform neuronal population, a method that does not involve the formation of EBs having the ability to differentiate into the three germ layer lineage is preferred. Also, considering application to human regenerative medicine, differentiation induction is performed in the absence of other biological materials (eg, feeders such as stromal cells and amniotic matrix) in order to avoid the risk of pathogen contamination and rejection. It is preferable. Furthermore, in biologically derived components (eg, serum) whose components are unknown, substances that have posterior activity for neural differentiation and subsequent region-specific differentiation induction (eg, BMP, LIF, Wnt, TGF-β) Therefore, it is preferable to induce differentiation using a chemical composition medium (that is, a limited medium) with a known component. Therefore, in a preferred embodiment of the present invention, a serum-free monolayer adhesion culture method, SFEB method, NSS method or the like is used.

As the basal medium in this differentiation-inducing step (b), the basal medium described above for maintenance and proliferation of pluripotent stem cells can be used similarly. Preferably, a serum-free medium, such as a serum-free medium supplemented with KSR at a concentration of 1-20%, or a serum-free medium supplemented with insulin and transferrin (for example, N2B27, CHO-S-SFM II (manufactured by GIBCO BRL) ), Hybridoma-SFM (GIBCO BRL), eRDF Dry Powdered Media (GIBCOBRL), UltraCULTURE ^TM (BioWhittaker), UltraDOMA ^TM (BioWhittaker), UltraCHO ^TM (BioWhittaker), UltraMDCK ^TM (BioWhittaker) , ITPSG medium (Cytotechnology, 5, S17 (1991)), ITSFn medium (Proc. Natl. Acad. Sci. USA, 77, 457 (1980)), mN3 medium (Mech. Dev. 59, 89 (1996) The medium may contain other components such as amino acids, pyruvic acid, 2-mercaptoethanol, cytokines, growth factors, and the like at appropriate concentrations as necessary.

ES cells can be easily expressed by removing differentiation inhibitors (eg, serum, LIF, BMP, Wnt, TGF-β, extracellular matrix, feeder cells) from the culture medium for maintenance and proliferation of pluripotent stem cells. An ectodermal cell and / or a differentiation inducer to a nervous system cell, for example, a Wnt inhibitor such as Dkk-1, in order to obtain a uniform nerve cell with higher differentiation induction efficiency, although it can differentiate into a progenitor cell (Nature Biotechnology, 20, 1240-1245 (2002)), Nodal inhibitors such as Lefty-A, BMP inhibitors such as BMPRIA-Fc and Noggin (Nature, 376, 333-336 (1995)), NGF (Biochem. Biophys. Res. Commun., 199, 552 (1994)), retinoic acid (Dev. Biol., 168, 342 (1995); J. Neurosci., 16, 1056 (1996)), FGF (Genes & Development, 15 , 3023-8 (2003)), IGF (Genes & Development, 15, 3023-8 (2003)) and the like can be added to the medium.
In addition, human pluripotent stem cells such as human ES cells are highly sensitive to mechanical and chemical stimuli.For example, when they are dissociated into single cells by normal trypsin treatment, their adhesion is significantly reduced and apoptosis is caused. Wake up. For this reason, human pluripotent stem cells cannot be stably cultured unless they are treated as a population of about several tens of cells, which prevents efficient differentiation induction. In order to suppress apoptosis due to the dispersion of the human pluripotent stem cells, it is preferable to treat the pluripotent stem cells with a ROCK inhibitor during the dispersion. Examples of ROCK inhibitors include Y-27632 (eg, Mol. Pharmacol. 57, 976-983 (2000); Methods Enzymol. 325,273-284 (2000)), Fasudil / HA1077 (eg, Nature 389: 990- 994 (1997)), H-1152 (eg, Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (eg, Cancer Chemother Pharmacol. 52 (4): 319-324 (2003)). ) And their derivatives.

As the incubator in this differentiation induction step (b), the incubator described above for maintenance and proliferation of pluripotent stem cells can be used similarly. In the case of the monolayer adhesion culture method, the incubator is preferably precoated with, for example, collagen, gelatin, matrigel, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.

In the culture, the Hes1 inactivated PSC has a cell density of, for example, about 0.5 to about 5 × 10 ⁴ cells / cm ² , preferably about 1 to about 3 × 10 ⁴ cells / cm ^2. For example, in an atmosphere of about 1 to about 10%, preferably about 2 to about 5% CO ₂ concentration in a CO ₂ incubator at about 30 to about 40 ° C., preferably about 37 ° C., about 2 -About 14 days, preferably about 4 to about 10 days.

Confirmation of whether or not the cultured cells obtained in this way have differentiated into uniform neurons uses RT-PCR, Northern blot analysis, immunostaining, immunoblot analysis, FACS, etc. , Nestin-1, Sox1, Mash1, etc.) and / or expression of neuronal markers (eg, Tuj-1, GAD, TH, etc.) can be used as indicators.

The cell population obtained by the differentiation inducing step (b) contains more uniform neurons than those obtained by the conventional method, but the cells are used as a therapeutic agent for diseases based on, for example, nervous system cells. For example, it is desirable to increase the purity of nerve cells prior to use.
Any known cell separation and purification method can be used as a method for increasing cell purity. For example, a method using a flow cytometer (for example, Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory). (1988), Monoclonal Antibodies: principles and practice, Third Edition, Acad. Press (1993), Int. Immunol., 10, 275 (1998)), panning methods (eg Monoclonal Antibodies: principles and practice, Third Edition, Acad. Press (1993), Antibody Engineering, A Practical Approach, IRL Pressat Oxford University Press (1996), J. Immunol., 141, 2797 (1988)), Cell fractionation method using density difference of sucrose concentration (For example, see the tissue culture technique (third edition), Asakura Shoten (1996)).

As another method for increasing the purity of nerve cells, there is a method of culturing the cells obtained by the differentiation induction step (b) in a medium containing an anticancer agent. Thereby, cells in an undifferentiated state can be removed, and tumor formation after transplantation can be prevented.
Here, examples of the anticancer agent include mitomycin C, 5-fluorouracil, adriamycin, ara C, and methotrexate. These anticancer agents are preferably used at concentrations that are more cytotoxic to cells that are undifferentiated than those that have undergone neuronal differentiation. For example, there can be mentioned a method of culturing cells for several hours, preferably 2 hours, using a medium containing these anticancer agents in a living body at a concentration of 1/100 times the concentration described in the Japanese Pharmacopoeia.

(C) Use of nerve cell derived from pluripotent stem cell The nerve cell obtained by the method of the present invention can be used for various purposes. For example, iPS induced using somatic cells collected from a patient who has a disorder based on a disorder of nervous system cells (hereinafter, also simply referred to as “neurological disorder”) or from another person who has the same or substantially the same type of HLA. Stem cell therapy by autologous or allogeneic transplantation in which nerve cells differentiated from cells are transplanted into the patient to regenerate nerves becomes possible. Furthermore, neuronal cells differentiated from patient-derived iPS cells are more likely to reflect the actual state of the neuronal cells in the patient's body than the corresponding existing cell lines. And in vitro evaluation system for toxicity. Furthermore, it can be preferably used as a tool for pathological research of neurological diseases whose cause has not been elucidated.

Hereinafter, the use of the pluripotent stem cell-derived nerve cell of the present invention in regenerative medicine will be described in detail. Examples of diseases that can be treated by the nerve cells include, for example, Parkinson's disease, spinocerebellar degeneration, Huntington's disease, Alzheimer's disease, ischemic brain disease (for example, cerebral infarction), epilepsy, brain trauma, etc. Disorders or disorders caused by telencephalic neurons; diseases based on cerebellar neurons, such as spinocerebellar degeneration, alcoholic cerebellar degeneration, cerebellar trauma; spinal cord injury, motor neuropathy, neurodegenerative disease, retinal pigment Examples include, but are not limited to, degeneration, age-related macular degeneration, inner ear deafness, multiple sclerosis, amyotrophic lateral sclerosis, and diseases caused by neurotoxin disorders.

As nerve cells for the treatment of hereditary neurological diseases such as Huntington's disease, nerve cells that have been induced to differentiate from pluripotent stem cells derived from others who have the same or substantially the same HLA type as the patient are preferred. used. Here, the type of HLA is “substantially the same” means that the transplanted cells are transplanted when cells obtained by inducing differentiation from iPS cells derived from the somatic cells are transplanted into a patient by using an immunosuppressant or the like. This means that the HLA types match to the extent that they can be engrafted. In human regenerative medicine, it is not easy to obtain human ES cells with the same or substantially the same type of HLA, so human iPS cells should be used as pluripotent stem cells for inducing nerve cells. Is preferred.

In another embodiment, nerve cells differentiated from iPS cells derived from the patient's own somatic cells can be used as nerve cells for the treatment of hereditary neurological diseases. In this case, since the iPS cells derived from the patient's somatic cells have an abnormality in the causative gene, a normal gene is introduced into the iPS cells. Adeno-associated virus (AAV) vectors are said to be suitable for gene transfer into neurons, but this vector has only a capacity of about 4.5 kb, so the full-length gene cannot be transferred depending on the size of the gene to be transferred. There is a case. However, in the case of iPS cells, retrovirus / lentivirus has the highest transfer efficiency, and since full-length genes can be transferred by artificial chromosomes, there are advantages that gene therapy options are expanded. Alternatively, the mutation site of the causative gene can be repaired using the endogenous DNA repair mechanism or homologous recombination of iPS cells. That is, a chimeric RNA / DNA oligonucleotide (chimeraplast) having a sequence with a normal mutation site is introduced and bound to a target sequence to form a mismatch, and an endogenous DNA repair mechanism is activated to induce gene repair. Alternatively, gene repair can be performed by introducing a single-stranded DNA having 400-800 bases homologous to the mutation site to cause homologous recombination. A normal nerve cell derived from the patient can be produced by inducing differentiation of the iPS cell obtained by repairing the disease-causing gene thus obtained into the nerve cell through the steps (a) and (b). it can. Alternatively, for polyglutamine diseases such as Huntington's disease, gene therapy that suppresses the production of abnormal proteins by the RNAi method has been proposed, and by introducing a vector that expresses siRNA against the causative gene into patient-derived iPS cells. Thus, patient-derived iPS cells in which abnormal protein accumulation is suppressed can be obtained and differentiated into nerve cells through the method of the present invention.

On the other hand, in the case of non-hereditary neurological diseases, nerve cells differentiated from iPS cells derived from the patient's own somatic cells may be normal cells and can be used for transplantation to patients as they are. There is a case.

In many neurological diseases, a specific cell type is specifically dropped. Therefore, it is also preferable to induce the necessary nervous system cells according to the target disease and then transplant the cells. For example, in Parkinson's disease, dopaminergic neurons are in amyotrophic lateral sclerosis, motor neurons are in Huntington's disease, GABAergic neurons are in cerebellar degeneration, Purkinje cells are in cerebral infarction Cerebral nerves and oligodendrocytes in the case of spinal cord injury, oligodendrocytes in the case of multiple sclerosis, cerebral nerves in the case of Alzheimer's disease, retinal neuronal cells in the case of retinitis pigmentosa Listed as cells.
It is possible to induce various neurons by causing the anteroposterior axis / dorsal ventral inductive factor to act at appropriate timing. For example, dopaminergic neurons are produced by, for example, the SDIA method and the 5-step method (PNAS, 101, 12543-8 (2004)), and motor neurons are produced by, for example, the SDIA method using retinoic acid, sonic hedgehog (Shh), GDNF, By means of the action of BDNF and ascorbic acid (Stem Cells, 25, 1931-9 (2007)), cerebral nerves, for example, by the SFEB method (Nature Biotechnol., 25, 681-6 (2007)), oligodendrocytes are For example, retinoic acid and EGF are allowed to act (J. Neurosci., 25, 4694-705 (2005)), and retinal neurons are allowed to act, for example, by Dkk1, IGF-1, and Noggin (PNAS, 103, 12769-74 (2006)), each can be guided.

The nerve cell of the present invention is produced as a parenteral preparation, preferably a parenteral preparation such as an injection, suspension, instillation, etc. by mixing with a pharmaceutically acceptable carrier according to a conventional method. Examples of pharmaceutically acceptable carriers that can be included in the parenteral preparation include isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants. An aqueous liquid for injection can be mentioned. The agent of the present invention includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum albumin, Polyethylene glycol, etc.), preservatives, antioxidants and the like.
When the agent of the present invention is formulated as an aqueous suspension, nerve cells may be suspended in the aqueous solution so as to be about 1.0 × 10 ^{6 to} about 1.0 × 10 ⁷ cells / mL.
Since the thus obtained preparation is stable and has low toxicity, it can be safely administered to mammals such as humans. The administration method is not particularly limited, but is preferably injection or drip administration, and examples include intravenous administration, intraarterial administration, and local administration of the affected area. The dose of the agent of the present invention varies depending on the administration subject, treatment target site, symptom, administration method, etc., but usually in Parkinson's disease patients (weight 60 kg), for example, once in the case of intravenous injection Conveniently, about 1.0 × 10 ^{5 to} about 1 × 10 ⁷ cells per dose are administered about 1 to about 2 weeks, about 4 to about 8 times.

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

[Materials and methods]
ES cell lines, culture conditions, and quantification of real-time imaging oscillation expression and Hes1 knockdown include the mouse ES cell MG1.19 cell line (Proc. Natl. Acad. Sci. USA 92, 1292 (1995), Nature 423, 541 (2003)). The TT2 cell line was used for genetic manipulation.
MG1.19 and TT2 cell lines are 10% (MG1.19) or 15% on gelatin coated plates (MG1.19) or mouse embryo fibroblast (MEF) feeder cell layer (TT2). (TT2) was maintained in DMEM medium supplemented with FCS, L-glutamine, non-essential amino acids, sodium pyruvate, 2-mercaptoethanol and 1,000 U / ml LIF.
Prior to performing the differentiation assay and the microarray assay, the suspension cells were subjected to suspension culture for 30 minutes after trypsin treatment twice to remove feeder cells. Nerve differentiation induction was performed on N2B27 medium (Methods Enzymol. 365, 327 (2003)).
For real-time imaging, ES cells were cultured in an ES medium supplemented with 1 mM luciferin on a glass dish. Fluorescence imaging was performed at 20 minutes or 10 minutes exposure, and binning processing was performed with 8 × 8 or 4 × 4 pixels in order to increase the SN ratio. Strong white dots are derived from cosmic rays. Images were collected from a CCD camera and analyzed with IMAGE-PRO as described in Proc. Natl. Acad. Sci. USA 103, 1313 (2006).

Measurement of mRNA and protein half-life For the measurement of mRNA half-life, actinomycin D (5 μg / ml) was added to the mouse ES cell MG1.19 cell line 30 minutes after the medium was changed (t = 0). added. Relative mRNA levels were quantified by real-time PCR using GAPDH as an internal control.
For measurement of protein half-life, cycloheximide (20 μM) was added to the mouse ES cell MG1.19 cell line 2 hours after the medium was changed (t = 0). The peak of Hes1 protein expression reached its maximum at t = 5 minutes. Relative expression levels were determined by Western blot.

Linearize the nuclear localization signal (NLS) of the Hes1 reporter cell line and the luc2 gene linked to two ubiquitins (Promega) and the reporter plasmid carrying the puromycin resistance gene (Ub-Ub-NLS-luc2) Electroporated into the MG1.19 cell line. The luciferase has a half-life of about 10 minutes in NIH3T3 cells.
After selecting cells with puromycin (2 μg / ml), cells having the plasmid cells were further selected based on luminescence. Of the 192 cell lines selected, 6 cell lines were used for single cell imaging.

Preparation of Hes1 knockout cells In one Hes1 allele, the ATG codon of the Hes1 gene was deleted, and a Ub-luciferase gene was inserted into the translation start site of Hes1 (FIG. 6A). In the other Hes1 allele, a loxP sequence was introduced at both ends of the Hes1 gene so as to sandwich the second exon to the fourth exon of the Hes1 gene (FIG. 6B). Homologous recombinants (121 and 152 lines) were selected by Southern plot analysis (FIGS. 6C and D). A Cre recombinase was allowed to act on these homologous recombinants to excise the second to fourth exons of the Hes1 gene to prepare Hes1-null cell lines (K3, K9, K57) (FIGS. 6E and F).

Preparation of Hes1 Constitutively Expressing ES Cells The targeting construct shown in FIG. 7A was knocked into the Rosa26 locus of mouse ES cells, and the integration of colonies selected in the presence of G418 was confirmed by Southern blot analysis (FIG. 7B). Cre recombinase was allowed to act on 21 lines of cells, the Neo expression cassette was excised, and GFP positive colonies were picked up to obtain Hes1 constitutive expression cell lines (R5, R6) (FIG. 7C). The constitutive expression of Hes1 was confirmed by immunostaining (FIG. 8A) and immunoblot analysis (FIG. 8B, C).

Hes1 knockdown (1) ES cells Polyomavirus ori (Proc. Natl. Acad. Sci. USA 92, 1292 (1995)) and puromycin resistance gene, 7SK promoter (Proc. Natl. Acad. Sci. USA) 104, 11292 (2007)), a plasmid vector encoding an shRNA sequence was prepared and introduced into mouse ES cell strain MG1.19 by lipofection. Transfected cells were selected on puromycin-containing medium.
The target nucleotide sequences used for the generation of Hes1 knockdown ES cells KD1 and KD2 are as follows.
KD1: 5'-GCCAATTTGCCTTTCTCATCC-3 '(SEQ ID NO: 7)
KD2: 5'-GTAGAGAGCTGTATTAAGTGA-3 '(SEQ ID NO: 8)
A random sequence was used as a control.
(2) iPS cell A lentiviral vector encoding shRNA and neomycin resistance gene is prepared, introduced into human iPS cell lines (iPS (IMR90) -1; WiCell and 253G1; RIKEN BRC), and selected in a medium containing G418. went.
The target nucleotide sequences used for the preparation of Hes1 knockdown iPS cells iPS-KD1 and iPS-KD2 are as follows.
iPS-KD1: 5'-GACCATGCACTATATTTGTAT-3 '(SEQ ID NO: 9)
iPS-KD2: 5'-GCATCTGAGCACAGAAAGTCA-3 '(SEQ ID NO: 10)
A random sequence was used as a control.

Induction of neural differentiation from iPS cells Induction of neural differentiation from human iPS cell lines (iPS (IMR90) -1 and 253G1) was performed according to the protocol of Gerrard L et al. (2005) Stem Cells 23: 1234-1241. . That is, the human iPS cell line was maintained in 5 ng / mL bFGF-added Prime ES culture mdeium (ReproCELL) on a plate seeded with mitomycin C-treated MEF. Cells that have reached confluence are treated with a cell dissociation solution (CTK: 0.25% Trypsin, 1mg / ml Collagenase IV, 20% KSR, 1mMCaCl2 / PBS) at 37 ° C for 3 minutes. After removing the cell dissociation solution, Separated by pipetting. 40 [mu] m, using a cell strainer of 70μm were collected cell masses having a diameter of 40-70μm, were seeded such that 10000 cells / cm ² in culture dishes polylysine / laminin coated, 100 ng / mL mouse recombinant Noggin containing or not The cells were cultured in N2B27 medium containing 7-14 days.

Microarray analysis GeneChip Mouse Genome430 2.0Array (Affimetrix) was used for microarray analysis. After removing feeder cells, total RNA was recovered from two ES cell lines and microarray analysis was performed. Data were analyzed with GCOS (Affimetrix) and normalized with parent cell values with Gene Spring (Agilent Technologies). The average of the two cell lines is shown in FIG. 9B and Table 1.
In ChIP-chip analysis, as a negative control, two types of Hes1 knockdown cells were mixed, and rabbit anti-Hes1 antibody was used as described in Biochem. Biophys. Res. Commun. 372, 142 (2008). Sedimentation was performed. Next, ChIP samples were amplified, hybridized with mouse promoter 1.0R array and mouse tiling 2.0R array (Affimetrix) and analyzed as described in Nature 451, 796 (2008).

Real-time PCR, immunostaining, and immunoblotting Quantification by real-time PCR and Western blot was performed as described in Proc. Natl. Acad. Sci. USA 104, 11292 (2007) and EMBO J. 18, 1192 (1999). In real-time PCR analysis, a standard curve was prepared using a cDNA mixed sample for each primer. Quantitative values of RNA and protein were normalized with the corresponding values of GAPDH and actin RNA and protein. Quantitative analysis was performed at least three times, and the results were shown with standard errors.
For immunohistochemistry, as shown below, cells were fixed with 4% PFA, blocked with 0.1% Triton-2% skim milk in PBS, and stained with the specific antibodies shown below.

Single cell quantitative PCR
One cell of wild-type mouse ES cell strain MG1.19 was picked randomly with a glass capillary mouse pipette. cDNA was prepared and amplified using the method described in Nature Protocols 2, 739 (2007). After amplifying the cDNA of one cell, quantitative PCR was performed using specific primers.
As described in Nucleic Acids Research 34, e42 (2006) and Development 135, 3113 (2008), for all primer pairs, the coefficients A and B were measured using the plasmid or mouse genome, and using these coefficients, The copy number of each gene was calculated and normalized using spike RNA Lys (1000 copies per cell).

The following antibodies were used for antibody western blotting: rabbit anti-Hes1 antibody (received from Dr. Sudo), goat anti-Oct3 / 4 antibody (N-19) and anti-p57 antibody (M-20) (Santa Cruz Biotechnology), Rabbit anti-actin antibody (SIGMA, A2066) and rabbit anti-cleaved Notch1 antibody (NICD) (Cell Signaling Technology).
Immunohistochemistry and chromatin immunoprecipitation include the following: rabbit anti-Tuj-1 antibody (Covance), mouse anti-Nestin antibody and anti-Oct3 / 4 antibody (BD Pharmingen), and rabbit anti-Hes1 produced in the present invention. Antibody was used.

Preparation of rabbit anti-Hes1 antibody Hes1 protein fused to maltose binding protein (MBP) is purified, injected into rabbits, and anti-MBP-Hes1 antibody is purified from serum by affinity binding. Only MBP antibody was absorbed. Recombinant MBP and MBP-Hes1 fusion proteins were purified as described in Biochem. Biophys. Res. Commun. 372, 142 (2008).

Secondary antibodies for secondary antibody fluorescence labeling were Alexa488-labeled anti-rabbit secondary antibody and anti-mouse secondary antibody, and Alexa594-labeled anti-rabbit secondary antibody and anti-mouse secondary antibody (Molecular Probes). .

Construction of Dll1 reporter and Gadd45g reporter The promoter region of pDll1-Ub1-Luc described in Neuron 58, 52 (2008) was replaced with a 6 kb Dll1 promoter having a Hes1 binding region sequence cloned from the mouse genome to produce a Dll1 reporter plasmid. . In addition, a 3-kb Gadd45g promoter having a Hes1 binding site, and 5'-UTR and 3'-UTR sequences of Gadd45g were cloned from the mouse genome, and the Gadd45g promoter and 5'-UTR were upstream of Ub-NLS-luc2. The Gadd45g reporter plasmid was constructed by connecting 3′-UTR of Gadd45g downstream. The lentiviral plasmid pCSII-EF-MCS was treated with AgeI to remove the EF promoter, and the Gadd45g reporter gene was subcloned. Lentiviral vectors were constructed according to the methods described in Methods in Molecular Biology 246, 429 (2004), Methods Enzymol. 420, 64 (2006) and Methods Enzymol. 420, 49 (2006).
The Dll1 reporter plasmid was introduced into mouse ES cell strain MG1.19 at MOI = 20-50 by Lipofectamine2000 (Invitrogen) and the Gadd45g reporter gene was introduced by lentivirus (Methods Enzymol. 420, 64 (2006)).

Spectral analysis of expression time was performed using the maximum entropy method (MEM) proposed by Burg (Modern Spectral Analysis (IEEE press, Newyork, 1978)) to estimate the period of Hes1 oscillation.

[result]
Reference Example 1 Confirmation of Hes1 Expression in ES Cells Hes1 expression in NIH3T3 cells (lane 1), MEF (lane 2) and mouse ES cells (lanes 3 and 4) was examined (FIG. 1A). Each cell extract (20 μg) was subjected to Western blot using anti-Hes1 antibody and anti-actin antibody. Actin was used as a loading control. As a result, expression of Hes1 was observed in ES cells. Next, the expression of Hes1 protein and mRNA in ES cells under various culture conditions was examined (FIG. 1B). Expression of Hes1 in ES cells was inhibited in the absence of LIF or BMP, but not in the presence of Notch signal inhibitor DAPT, and Notch signal did not regulate Hes1 expression in ES cells (FIG. 1C). ).

Reference Example 2 Observation of Oscillation of Hes1 Expression by Immunostaining and Real-Time Imaging Mouse ES cells were immunostained using fluorescently labeled anti-Hes1 antibody and anti-Oct3 / 4 antibody. The results are shown in FIG. In DAPI staining, all ES cells were stained with uniform fluorescence intensity, whereas in the immunostaining with anti-Hes1 antibody, the fluorescence intensity after immunostaining of each ES cell was not uniform (FIG. 2A). Next, expression of Hes1 in individual ES cells was observed over time using a reporter plasmid in which firefly luciferase was linked downstream of the Hes1 promoter. As a result, it was observed that luminescence of luciferase fluctuated with time even in one cell (A to C) (FIG. 2B). When the luminescence intensity of luciferase was measured and plotted for each cell, it was found that the expression of Hes1 in the cell was oscillating and that this oscillation was not synchronized between cells (FIG. 2C).

Reference Example 3 Half-life of Hes1 protein and mRNA, time course of Hes1 expression Cycloheximide (CHX) inhibited new protein synthesis and determined the half-life of Hes1 protein in ES cells. The half-life was 16 minutes. (FIG. 3A). Addition of the proteasome inhibitor MG132 markedly stabilized the Hes1 protein. When the half-life of Hes1 mRNA was also examined, it was about 46 minutes in ES cells, but about 10 minutes in C3H10T1 / 2 fibroblasts (FIG. 3B). When the time course of Hes1 expression in ES cells was examined, the expression levels of Hes1 mRNA and protein increased after medium replacement. Hes1 reporter activity increased prior to Hes1 mRNA and protein. Luminescence by luciferase appeared before Hes1 mRNA was expressed and inversely correlated with the expression of Hes1 protein (FIG. 3C).

Reference Example 4 Oscillation of Hes1 in daughter cells of ES cells It was observed whether the oscillation of Hes1 in ES cells was inherited by each daughter cell even after the ES cells were divided (FIG. 4). The ES cells divided into two daughter cells at time t = 400 (point of arrow in the figure). Three peak points are indicated by asterisks. Oscillation of Hes1 expression in ES cells was synchronized between post-mitotic daughter cells.

Reference Example 5 Spectral analysis of Hes1 expression in ES cells The expression of Hes1 in 5 ES cells was monitored and plotted on a chart (FIG. 5A, upper panel). Furthermore, the obtained data was Fourier-transformed using the maximum entropy method and plotted on the power spectrum (FIG. 5A, lower panel). The oscillation period was obtained by determining the peak frequency of the power spectrum. When the cycle (Y axis) in each cell (X axis) was plotted, it was roughly divided into a group of 3 to 5 hours and a group of less than 2 hours (FIG. 5B). The latter seems to be due to irregular period or experimental noise. The cycle of 3-5 hours in ES cells was longer than other cell types (2-3 hours). Since the half-life of Hes1 protein in ES cells is about 16 minutes, which is equivalent to that of fibroblasts, the half-life of mRNA is about 4.5 times longer than that of fibroblasts (FIGS. 3A and B). It is thought that it contributes to the long periodicity in ES cells.

Example 1 Preparation of Hes1-inactivated ES cells and maintenance of ES cell properties As described in [Materials and Methods] above, Hes1 knockout cells (K3, K9, K57), Hes1 constitutively expressing cells (R5, R6) and Hes1 knockdown cells (KD1, KD2) were prepared. Each of these cells (excluding K57) was subjected to Western blotting with an anti-Hes1 antibody to verify the expression level of Hes1. The results are shown in FIG. 9A. In Hes1 knockout cells (lanes 2 and 3) and Hes1 knockdown cells (lanes 8 and 9), no band corresponding to Hes1 was detected, confirming that Hes1 was completely inactivated. On the other hand, Hes1 constitutive expression cells (lanes 5 and 6) expressed Hes1 at a higher level than wild-type ES cells.
Next, the expression of ES cell marker gene in Hes1 knockout cells (K3, K9, K57) and Hes1 constitutive expression cells (R5, R6) was examined by quantitative PCR. As a result, for Nanog, Oct4, Sox2, and Rex1, all cells showed the same level of expression as wild-type ES cells (FIG. 10A). Moreover, all the cells were able to form three germ layers and maintained pluripotency. That is, it was shown that inactivation of Hes1 does not affect the maintenance and proliferation of ES cells.

Example 2 Identification of downstream genes whose expression is changed by Hes1 inactivation Using a microarray, mRNA between Hes1 knockout cells (K3, K9), Hes1 constitutively expressed cells (R5, R6) and wild type ES cells Expression was comprehensively compared. As a result, we found 36 genes that showed more than twice the expression in Hes1 knockout cells compared to the expression in Hes1 constitutively expressing cells (Table 1). Of these, 7 genes shown in FIGS. 9B and 10B were identified as targets of Hes1 by real-time PCR.

In addition, chromatin immunoprecipitation using anti-Hes1 antibody was performed, and the DNA precipitate was subjected to oligonucleotide tiling array (ChIP-chip) to directly identify the target gene of Hes1. As a result, 506 Hes1 binding regions were identified. Among them, genes corresponding to the top 50 regions of signal intensity are shown in Tables 2-1 and 2-2. Among the 7 genes identified by the microarray analysis, 5 genes of Gadd45g, Dll1, Lef1, Jag1, and Crabp2 were also identified by ChIP-chip analysis (FIG. 9C).

Example 3 Oscillation of downstream genes of Hes1 Among the downstream genes of Hes1 identified in Example 2, two genes Dll1 and Gadd45g, which had the highest signal intensity in ChIP-chip analysis, were real-time using a ubiquitin fusion reporter. The expression kinetics were further analyzed by imaging. The results are shown in FIG. The expression levels of the Dll1 gene and the Gadd45g gene were also found to oscillate in the ES cells as in the Hes1 gene (FIG. 11A). In order to examine the relationship between Hes1 expression and Dll1 and Gadd45g expression, 32 ES cells were randomly picked up, and single-cell quantitative PCR was performed using cDNA collected from each cell as a template. These ES cells were classified into 3 groups according to the copy number (log ₁₀ ) of Hes1 (FIG. 11B), and compared with the expression of Dll1 and Gadd45g, the expression of these downstream genes was also observed in the Hes1 low expression cell group. The expression of the downstream gene was low in the low Hes1 highly expressing cell group, and it was found that Hes1, Dll1, and Gadd45g oscillate in the same phase in each ES cell (FIG. 11C). From this, it was considered that the expression of Hes1, Dll1 and Gadd45g was suppressed when the Hes1 protein level was high, and that the expression of these genes was activated when the Hes1 protein level was low. On the other hand, the p57 gene whose expression was increased by Hes1 knockout by microarray analysis showed an expression pattern different from Dll1 and Gadd45g (FIG. 11C). In addition, among the ES cell-specific genes, Nanog expression varied in the same phase as Hes1 (FIG. 11C).

Example 4 Differentiation Induction of Neurons from Hes1-Inactivated ES Cells Hes1 knockout cells (K9, K57), Hes1 constitutive expression cells (R5, R6) and wild-type ES cells were treated with LIF-free medium without feeder cells. Cultured in the presence (known as the condition that induces differentiation in all three germ layers), quantified by real-time PCR, the expression of ectodermal / neural marker Mash1, mesodermal marker brachyury, and endoderm marker Gata4 gene did. In wild-type ES cells, all marker genes were activated within 4 days after LIF removal, whereas in Hes1 constitutively expressing cells, none of the marker genes was significantly upregulated, indicating sustained Hes1 expression. It was suggested that differentiation was inhibited (FIG. 12A). In contrast, upregulation of Mash1 was more prominent in Hes1 knockout cells than in other cells (FIG. 12A). In Hes1 knockout cells, another ectoderm marker Cxcl12 is also activated, and these results indicate that ectoderm formation is promoted in the absence of Hes1.

Example 5 Neuronal differentiation induction from Hes1-inactivated ES cells under neural differentiation conditions Hes1 knockout cells (K9, K57), Hes1 constitutive expression cells (R5, R6) and wild-type ES cells Cultured under conditions. In Hes1 constitutively expressed cells, the decrease in Oct4 expression was delayed and large colonies were maintained even after 4 days of induction of differentiation. Mash1, neural progenitor marker Nestin, and neuronal cell marker βIII-tubulin (Tuj-1) Expression was inhibited (FIGS. 12B, C). These Hes1 constitutively expressing cells were delayed in the upregulation of Fgf5, which is an early differentiation marker of ES cells, expressed brachyury continuously (FIG. 13), and took a flat form after 6 days of induction of differentiation (FIG. 13). 12C). That is, it was shown that constitutive expression of Hes1 delays differentiation and prioritizes differentiation into mesoderm even under neural differentiation conditions. In contrast, inactivation of Hes1 enhanced expression of Mash1, Nestin, Tuj-1 (FIGS. 12B, D). When we examined the expression of Hes1 target gene and Notch signal target gene Hes5 in each ES cell during neural differentiation induction, Hes1 knockout cells showed a marked increase in Hes5 gene expression after differentiation induction, and the intracellular domain of Notch ( Increased production of (NICD) showed that Notch signal was activated by Hes1 inactivation (FIG. 14). Wild-type ES cells differentiated into neurons only at random even under neuronal differentiation conditions, while Hes1 knockout cells uniformly differentiated into neurons at earlier timing (FIGS. 12D and E).

Example 6 Induction of Neuronal Differentiation from Hes1 Knockdown iPS Cells under Neural Differentiation Conditions Hes1 knockdown human iPS cells (iPS-KD1, iPS-KD2) and control iPS cells (non-specific shRNA-introduced cells) The cells were cultured under neural differentiation conditions. In iPS-KD1 and iPS-KD2, the expression of Hes1 was suppressed by about 60 to 80% compared to the control (FIG. 15A). On the 7th, 10th, and 14th days after differentiation induction, the expression of the neural stem cell marker Sox1 gene and the neural cell marker βIII-tubulin (Tuj-1) gene was quantified by real-time PCR. As a result, it was found that the expression of Sox1 and Tuj-1 was significantly increased in Hes1 knockdown cells compared to control cells (FIGS. 15B and C). That is, it became clear that iPS cells also promote neuronal differentiation by Hes1 knockdown, similar to ES cells.

The present invention is an excellent method capable of producing uniform nerve cells in a short period of time from pluripotent stem cells such as ES cells and iPS cells, and is expected to be applied clinically in the field of regenerative medicine. For patients with neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease, neurons produced uniformly from pluripotent stem cells by in vitro culture according to the method of the present invention It is useful for use in “neural cell transplantation” in which transplantation is performed at the site of injury of a neurodegenerative disease.

Although the present invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments can be modified. The present invention contemplates that the present invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.

The contents of all publications, including the patents and patent application specifications mentioned herein, are hereby incorporated by reference herein to the same extent as if all were expressly cited. .

This application is based on Japanese Patent Application No. 2009-168045 filed on Jul. 16, 2009, the entire contents of which are included in this specification.

Claims (10)

1. A method for producing nerve cells from pluripotent stem cells, which comprises inhibiting Hes1 expression or function in pluripotent stem cells.
2. The method of Claim 1 including the following steps.
   (A) a step of contacting a pluripotent stem cell with a substance that inhibits the expression or function of Hes1 (b) a step of culturing the cell under conditions for inducing differentiation into a neuronal cell
3. The method according to claim 2, wherein the substance that inhibits or functions Hes1 expression is any of the following (a) to (h):
   (A) antisense nucleic acid against nucleic acid encoding Hes1 (b) siRNA or miRNA for RNA encoding Hes1 or precursor thereof (c) ribozyme nucleic acid for RNA encoding Hes1 (d) Hes1 inhibitor or encoding it Nucleic acid (e) Hes1 decoy nucleic acid (f) Dominant negative mutant of Hes1 or nucleic acid encoding it (g) Antibody to Hes1 or nucleic acid encoding it (h) Nucleic acid knocking out the Hes1 gene by homologous recombination
4. The method according to claim 2 or 3, wherein the step (b) is performed under one or more conditions selected from the following (a) to (c).
   (A) Does not induce embryoid body formation (b) Uses a limited medium (c) Does not use a feeder
5. The method according to any one of claims 1 to 4, wherein the pluripotent stem cells are ES cells or iPS cells.
6. The method according to any one of claims 1 to 5, wherein the pluripotent stem cell is derived from a human or a mouse.
7. Pluripotent stem cells into which nucleic acid factors that inhibit the expression or function of Hes1 are stably introduced.
8. A neuronal cell induced to differentiate from the pluripotent stem cell according to claim 7.
9. An agent for inducing differentiation of pluripotent stem cells into neurons, comprising any of the following substances (a) to (h):
   (A) antisense nucleic acid against nucleic acid encoding Hes1 (b) siRNA or miRNA for RNA encoding Hes1 or precursor thereof (c) ribozyme nucleic acid for RNA encoding Hes1 (d) Hes1 inhibitor or encoding it Nucleic acid (e) Hes1 decoy nucleic acid (f) Dominant negative mutant of Hes1 or nucleic acid encoding it (g) Antibody to Hes1 or nucleic acid encoding it (h) Nucleic acid knocking out the Hes1 gene by homologous recombination
10. RNA encoding Hes1, which targets a partial nucleotide sequence represented by nucleotide numbers 920 to 940 or 1229 to 1249 in the nucleotide sequence represented by SEQ ID NO: 1, or a sequence corresponding to the partial nucleotide sequence in a Hes1 gene other than mouse SiRNA or precursor thereof.

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