JPWO2017209290A1 - Production and use of pluripotent stem cell-derived motor neurons - Google Patents

Abstract

The present invention provides a method for producing motor neurons from pluripotent stem cells comprising the following steps:
(1) introducing a single Sendai virus vector into a pluripotent stem cell comprising a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2 and a nucleic acid encoding Isl1, and
(2) A step of culturing the pluripotent stem cells for 2 days or more.

Description

  The present invention relates to a method for producing motoneurons from pluripotent stem cells, a motoneuron inducer, the method, a drug screening method using motoneurons obtained by using the inducer, and the like.

  Amyotrophic lateral sclerosis (hereinafter also referred to as “ALS”) is the most common and severe form of motor neuron disease (hereinafter also referred to as “MND”) and causes progressive muscle weakness. Causes death within a few years. To date, a vast amount of knowledge about ALS has been reported, but the key mechanisms of this disease are still not fully understood.

  In recent years, the establishment of induced pluripotent stem cells (hereinafter also referred to as “iPS cells”) has provided a new approach to MND research and has led to the discovery of new drugs. Since the first generation of motor neurons (MN) derived from iPS cells of ALS patients in 2008, the establishment of a large number of motor neurons derived from ALS iPS cells has been reported (for example, see Non-Patent Document 1). Many of these methods rely on the principles of development and are based on the addition of various signaling molecules, such as retinoic acid (RA) and sonic hedgehog (Shh), to the medium. Changes in the combination of signaling molecules are required, and in some cases it can take as long as 4 weeks or more to differentiate into functional motor neurons.

  On the other hand, an attempt has been made to rapidly and efficiently differentiate pluripotent stem cells (PSCs) into motor neurons by exogenously introducing transcription factors involved in motor neuron differentiation. For example, Hester et al. Used nucleic acids encoding the transcription factors neurogenin 2 (Ngn2), islet-1 (Isl-1), and LIM / homeobox protein 3 (Lhx3) as human embryonic stem cells (ES cells) or iPS. It has been reported that differentiation into motor neurons was rapidly induced by introduction into cell-derived neural progenitor cells (Non-patent Document 2). Son et al. Reported that mouse and human fibroblasts were directly converted into motor neurons using 7 and 8 transcription factors including the above 3 transcription factors, respectively (Non-patent Document). 3). Furthermore, in 2013, Mazzoni et al. Reported that motoneurons were rapidly and efficiently obtained using mouse ES cells expressing Ngn2, Isl-1, and Lhx3 in a Dox-inducible manner (non-patented). Reference 4).

However, in these methods, the transgene is integrated into the host genome. Integration of vector genes into the host genome entails a risk of affecting host cell behavior. Hester et al. Use adenoviral vectors that rarely integrate into chromosomes for gene transfer into human ES cells, but under conditions that require severe selective pressure such as differentiation / dedifferentiation into target cells, In some cases, built-in events that should be very rare may become apparent. Moreover, in order to obtain the continuous expression of the transgene, a plurality of introduction operations are required (Non-patent Document 2).
Furthermore, in order to induce functional motoneurons in human cells, these methods still require 3 to 4 weeks (Non-Patent Documents 2 and 3), and different combinations of signaling factors 2 It is necessary to use media having three types of composition (Non-patent Documents 2 to 4).

Dimos JT. Et al., Science. 2008 Aug 29; 321 (5893): 1218-21. Hester ME. Et al., Mol Ther. 2011 Oct; 19 (10): 1905 -12. Son EY. Et al., Cell Stem Cell. 2011 Sep 2; 9 (3): 205-18. Mazzoni EO. Et al., Nat Neurosci. 2013 Sep; 16 (9): 1219-27.

  Accordingly, an object of the present invention is to provide a novel differentiation induction method and differentiation that can be differentiated from a pluripotent stem cell to a motor neuron in human cells without any risk of integration of an exogenous nucleic acid into the host genome. An inducing agent is provided, and using the method and agent, a homogeneous pluripotent stem cell-derived motor neuron (MND PSC-MN) that reproduces the MND phenotype is established, and the MND phenotype in the cell is established. It is to provide a more reliable screening method for MND therapeutic drug candidates using change as an index.

  In order to achieve the above-mentioned problems, the present inventors focused on Sendai virus (SeV) vectors that are not incorporated into the host cell genome in principle. First, the three transcription factors commonly used in the previous report ( Three types of SeV vectors each independently carrying a nucleic acid encoding Lhx3, Ngn2, and Isl-1) were constructed, and one or more of them were introduced into human iPS cells. As a result, it was revealed that motor neurons can be induced from iPS cells by introducing at least two factors, Lhx3 and Ngn2. This suggests that when the three factors are introduced with separate vectors, the resulting motor neuron population may contain motor neurons induced only by the two factors Lhx3 and Ngn2. The present inventors considered that there is a concern that the inhomogeneity may affect the performance as an in vitro MND model, for example, the phenotype of MND PSC-MN varies.

Therefore, the present inventors designed a single SeV vector (SeV-LNI) encoding Lhx3, Ngn2, and Isl-1 in order to obtain a more homogeneous pluripotent stem cell-derived motor neuron. When introduced into cells and human ES cells, the efficiency of motoneurons in the entire neuron increased dramatically, although the differentiation efficiency into neurons decreased compared to when the three factors were introduced with separate vectors. . That is, it was revealed that the differentiation behavior into motor neurons was more uniformly shown by ensuring the introduction of the three factors into pluripotent stem cells. Moreover, by culturing in a substantially single medium composition from the time of gene introduction, it was possible to differentiate into HB9-positive motor neurons within a very short period of time within 2 days after introduction. In addition, motor neurons derived from SePS-LNI from familial ALS patients and mouse models with mutations in the SOD1 or TDP-43 gene all showed the original ALS phenotype. The MND PSC-MN obtained by this method was proved to be useful as an in vitro model of MND.
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention is as follows.
[1] A method for producing motor neurons from pluripotent stem cells, comprising the following steps:
(1) introducing a single Sendai virus vector into a pluripotent stem cell comprising a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2 and a nucleic acid encoding Isl1, and
(2) A step of culturing the pluripotent stem cells for 2 days or more.
[2] The method according to [1], wherein the Sendai virus vector comprises a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2, and a nucleic acid encoding Isl1 in this order from 3 ′ side to 5 ′ side successively. .
[3] The method according to [1] or [2], wherein the culturing step is performed in a medium containing a single component motor neuron differentiation signal factor throughout the entire period.
[4] The method according to [3], wherein the motor neuron differentiation signal factor comprises retinoic acid, a sonic hedgehog (SHH) signal stimulator, and a neurotrophic factor.
[5] The method according to any one of [1] to [4], wherein the proportion of motor neurons in the total obtained neurons is 60% or more.
[6] The method according to any one of [1] to [5], wherein the pluripotent stem cells are derived from an animal exhibiting a motor neuron disease phenotype.
[7] The method according to [6], wherein the motor neuron disease is amyotrophic lateral sclerosis (ALS).
[8] The method according to any one of [1] to [7], wherein the pluripotent stem cells are ES cells or iPS cells.
[9] The method according to any one of [1] to [8], wherein the pluripotent stem cell is derived from a human or a mouse.
[10] The method according to [9], wherein the pluripotent stem cell is an iPS cell derived from an ALS patient.
[11] A motor neuron population obtained by the method according to any one of [1] to [10].
[12] A motor neuron population that reproduces the phenotype of motor neuron disease obtained by the method according to any one of [6] to [10].
[13] A Sendai virus vector comprising a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2, and a nucleic acid encoding Isl1.
[14] The vector according to [13], comprising a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2, and a nucleic acid encoding Isl1 in this order from 3 ′ side to 5 ′ side.
[15] A motor neuron differentiation inducer from pluripotent stem cells, comprising the vector according to [13] or [14].
[16] A screening method for a therapeutic drug candidate substance for motor neuron disease, comprising the following steps:
(1) contacting the test substance with the motor neuron population according to [12],
(2) measuring the degree of motor neuron disease phenotype in the cell population; and
(3) A step of selecting a test substance having an improved degree of the phenotype as a therapeutic drug candidate substance for motor neuron disease as compared with a case where the test substance is not contacted.
[17] The method according to [16], wherein the motor neuron disease is ALS.

According to the present invention, since a transgene or a vector-derived nucleic acid is not integrated into the host cell genome, there is no possibility that the behavior of the host cell will be affected by genomic disturbance. Furthermore, according to the present invention, since the three factors Lhx3, Ngn2 and Isl1 are introduced at a time, the introduction rate of each factor does not vary among cells, and differentiation was induced by the expression of the three factors. A homogeneous motor neuron population can be obtained. Therefore, the MND PSC-MN induced by the method of the present invention has a more reliable MND than that provided by the conventional method, which eliminates the problems of gene transfer and cell heterogeneity. It can be an in vitro model.
In addition, according to the present invention, human pluripotent stem cells can be differentiated from motor neurons in a very short period of time without substantially changing the medium composition during the culture. Excellent speediness.

FIG. 4 shows motor neuron induction by three separate Sendai virus vectors. (a) Outline of experimental procedure for producing motor neurons (MN) from HB9-EGFP knock-in human iPS cells using SeV-Lhx3, SeV-Ngn2 and SeV-Isl1. (b) Immunofluorescent staining of iPS cell-derived motor neurons for HB9 and ChAT (motor neuron markers), and Tuj1 and MAP2 (neuron markers) And merge with DAPI stained nuclei). Scale bar = 20 μm. (c) qPCR analysis of differentiated cells on day 0 and day 14 for motor neuron markers (HB9 and ChAT) and neuronal marker (MAP2). Student t test was used for statistical comparison. ^* p <0.05. Error bars are SEM, n = 3. It is a figure which shows the comparison between the combination of three SeV vectors. (a) Immunofluorescent staining of HB9 (upper) and Tuj1 (middle) of cells differentiated by various combinations of SeV-Lhx3 (L), SeV-Ngn2 (N), and SeV-Isl1 (I); Shows the merge of these and DAPI stained nuclei. Scale bar = 40 μm. (b) Percentage of HB9 positive (motor neurons; left) and Tuj1 positive (neurons; right) cells obtained with various combinations of transcription factors. Data were analyzed by one-way analysis of variance and Dunnette's post-comparison analysis. ^* p <0.05. Error bars are SEM, n = 3. It is a figure which shows the induction | guidance | derivation and functional analysis of a motor neuron by SeV-Lhx3-Ngn2-Isl1. (a) Outline of experimental procedure for generating motor neurons from HB9-EGFP knock-in human iPS cells using a single vector, SeV-Lhx3-Ngn2-Isl1 (SeV-LNI). (b) Immunofluorescent staining of cells induced by a single vector against HB9, Tuj1, MAP2 and ChAT (the upper and lower rows are the same field of view, and the rightmost column shows each stained image and DAPI-stained nucleus. And the percentage of HB9 positive (motor neurons; left) and Tuj1 positive (neurons; right) cells obtained. Error bars are SEM. (c) qPCR analysis of differentiated cells on day 0 and day 14 for HB9, ChAT and MAP2. Student t test was used for statistical comparison. ^* p <0.05. Error bars are SEM, n = 3. (d) Current clamp recording of spontaneous action potential. (e) Double immunostaining for GFP for motoneurons and α-bungarotoxin for acetylcholine receptors. Scale bar = 10 μm. It is a figure which shows the expression of the neuron marker and the motor neuron marker in the cell which differentiated from the human and mouse | mouth iPS cell. (ab) Immunofluorescent staining of (a) HB9 (upper) and Tuj1 (middle), (b) ChAT (upper) and Tuj1 (middle) in cells induced via SeV-LNI Shows merge with DAPI stained nuclei). Human iPS cell lines are derived from normal (control), SOD1-ALS patients, and TDP-43-ALS patients. The mouse iPS cell line is derived from a normal mouse (control), a mutant SOD1 transgenic mouse, and a mutant TDP-43 transgenic mouse. Scale bar = 20 μm. It is a figure which shows the phenotype of the motor neuron induced | guided | derived from SOD1-ALS and TDP-43-ALS iPS cell by SeV-L-N-I. (a) Motor neurons from control and SOD1-ALS mouse iPS cells immunostained for misfolded SOD1 and Tuj1. Scale bar = 10 μm. (b) Motor neurons from control and TDP-43-ALS mouse iPS cells were immunostained for TDP-43 and Tuj1. (c) Motor neurons from control and ALS (SOD1-ALS) patient iPS cells were immunostained for misfolded SOD1 and Tuj1. Scale bar = 10 μm. (d) Motor neurons from control and ALS (TDP43-ALS) patient iPS cells were immunostained for TDP-43 and Tuj1. Scale bar = 10 μm. It is a figure which shows the induction | guidance | derivation from a human ES cell to a motor neuron by SeV-LNI. (a) Outline of experimental procedure for creating motor neurons in H9 ES cells using a single vector, SeV-LNI. (b) Immunofluorescent staining showing the expression of motor neuron markers (HB9 and ChAT) and neuron markers (Tuj1 and MAP2) (the upper and lower rows are the same field of view, the rightmost is the nucleus stained with each stained image and DAPI stain) Shows merge with). Scale bar = 20 μm. (c) qPCR analysis of differentiated cells on day 0 and day 14 for motor neuron markers (HB9 and ChAT) and neuronal marker (MAP2). Student t test was used for statistical comparison. For day 0, ^* p <0.05. Error bars are SEM, n = 3.

<Method for producing motor neuron or nerve cell>
The present invention provides a method for producing motor neurons from pluripotent stem cells (hereinafter also referred to as “the production method of the present invention”). The method comprises the following steps:
(1) introducing a single Sendai virus vector into a pluripotent stem cell comprising a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2 and a nucleic acid encoding Isl1, and
(2) including a step of culturing the pluripotent stem cells for 2 days or more.

1. Pluripotent stem cell In the present invention, a pluripotent stem cell is a stem cell that has pluripotency that can be differentiated into all cells present in a living body and also has proliferative ability, and is not particularly limited. For example, embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), sperm stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem cells ( iPS cells), cultured fibroblasts, pluripotent cells derived from bone marrow stem cells (Muse cells), and the like. Preferred pluripotent stem cells are ES cells and iPS cells. The origin of the pluripotent stem cell is not particularly limited, and examples thereof include any animal that has been reported to establish any of the following pluripotent stem cells, preferably mammals, more preferably humans and mice.

(A) Embryonic stem cells (ES cells)
ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, the embryo after the morula, in the 8-cell stage of a fertilized egg, and have the ability to differentiate into any cell that constitutes an adult, so-called differentiation. And ability to proliferate by self-replication. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and then ES cell lines have also been established in primates such as humans and monkeys (JA Thomson). et al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55: 254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).

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

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

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

  Human ES cell lines are, for example, WA01 (H1) and WA09 (H9) from WiCell Research Institute, KhES-1, KhES-2 and KhES-3 are from Kyoto University Research Institute for Regenerative Medicine (Kyoto, Japan). ).

(B) Sperm stem cells Sperm stem cells are testis-derived pluripotent stem cells that are the origin of spermatogenesis. Like ES cells, these cells can be induced to differentiate into various types of cells, and have characteristics such as the ability to create chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. ( 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). It is capable of self-replication in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and by repeating subculture under the same culture conditions as ES cells, Stem cells can be obtained (Masatake Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 41-46, Yodosha (Tokyo, Japan)).

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

(D) Artificial pluripotent stem cells (iPS cells)
iPS cells can be produced by introducing a specific reprogramming factor into somatic cells in the form of DNA or protein, and have almost the same properties as ES cells, such as pluripotency and proliferation ability by self-replication, (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); International Publication WO 2007/069666).

  As used herein, the term “somatic cell” refers to any animal cell (preferably, a mammalian cell including a human) except a germ line cell such as an egg, oocyte, ES cell, or totipotent cell. Say. Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells. , Passage cells, and established cell lines. Specifically, somatic cells include, for example, (1) neural stem cells, hematopoietic stem cells, mesenchymal stem cells, tissue stem cells such as dental pulp stem cells (somatic stem cells), (2) tissue progenitor cells, (3) lymphocytes, epithelium Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, enterocytes, spleen cells, pancreatic cells (exocrine pancreas cells, etc.), brain cells, lung cells, kidney cells Examples thereof include differentiated cells such as fat cells.

  Further, from the viewpoint of preparing a model cell for motor neuron disease (MND), iPS cells may be produced using somatic cells derived from an MND patient. Here, “motor neuron disease (MND)” is a disease that causes degeneration of motor neurons, such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and bulbar spinal cord. Examples include muscular atrophy (SBMA). For example, ALS patient somatic cells include somatic cells with mutations in the SOD1 gene and TDP-43 gene, and more specifically, mutations in SOD1 genes such as G93A, G93S, H46R and L144FVX, and M337V, Q343R And mutations of TDP-43 gene such as A315T, but are not particularly limited thereto.

  The reprogramming factor is a gene specifically expressed in ES cells, its gene product or non-cording RNA, a gene that plays an important role in maintaining undifferentiation of ES cells, its gene product or non-cording RNA, or It may be constituted by a low molecular compound. Examples of genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 etc. are exemplified, and these reprogramming factors may be used alone or in combination. As combinations of reprogramming factors, WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO 2010/056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO 2010/111409, WO 2010/111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. 2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467 -2474, Huangfu D, et al. (2008), Nat Biotechnol. 26: 1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008) ), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11: 197-203, RL Judson et al., (2009), Nat. Biotech., 27: 459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106: 8912-8917, Kim JB, et al. 2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5: 491-503, Heng JC, et al. (2010), Cell Stem Cell. 6: 167-74 , Han J, et al. (2010), Nature. 463: 1096-100, Mali P, et al. (2010), Stem Cells. 28: 713-720, Maekawa M, et al. (2011), Nature. 474: 225-9. Is exemplified.

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

  In the form of a protein, the reprogramming factor may be introduced into a somatic cell by a technique such as lipofection, fusion with a cell membrane-permeable peptide (for example, TAT and polyarginine derived from HIV), or microinjection.

  On the other hand, in the case of DNA, it can be introduced into somatic cells by techniques such as vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, and microinjection. Examples of viral vectors include retroviral vectors and lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (WO 2010/008054) and the like. In light of the object of the present invention to provide iPS cell-derived motoneurons without integration of the transgene into the host chromosome, the iPS cells should also be established without integration of the reprogramming gene into the chromosome. For this purpose, the use of Sendai virus vectors, which are RNA cytoplasmic vectors that have no chromosomal integration in principle, or rare or relatively low-frequency adenovirus vectors or adeno-associated virus vectors, is suitable. However, it is not limited to these. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). As a plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). The vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. so that a nuclear reprogramming substance can be expressed. Selective marker sequences such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG, etc. Can be included. In addition, the above vector has a LoxP sequence before and after the introduction of the gene into a somatic cell in order to excise the gene or promoter encoding the reprogramming factor and the gene encoding the reprogramming factor that binds to it. May be.

  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, a transposon derived from lepidopterous insects (Kaji, K. et al., (2009), Nature, 458: 771-775, Woltjen et al., (2009), Nature, 458: 766-770, WO 2010/012077). In addition, the vector replicates without chromosomal integration and is present episomally, so that the origin and replication of lymphotrophic herpes virus, BK virus, and bovine papillomavirus The arrangement | sequence which concerns on may be included. Examples include EBNA-1 and oriP or Large T and SV40 ori sequences (WO 2009/115295, WO 2009/157201, WO 2009/149233, WO 2011/016588). In order to further increase the reprogramming efficiency, a separate plasmid that supplies EBNA-1 to the trans may be co-introduced separately from the episomal vector carrying the reprogramming gene (WO 2013/176233). Moreover, in order to simultaneously introduce a plurality of nuclear reprogramming substances, an expression vector for polycistronic expression may be used. For polycistronic expression, the gene coding sequence may be linked by an IRES or foot-and-mouth disease virus (FMDV) 2A coding region (Science, 322: 949-953, 2008 and WO 2009 / 092042, WO 2009/152529).

  In the case of RNA form, it may be introduced into somatic cells by techniques such as lipofection and microinjection, and RNA containing 5-methylcytidine and pseudoridine (TriLink Biotechnologies) is used to suppress degradation. (Warren L, (2010) Cell Stem Cell. 7: 618-630).

  As a culture solution for iPS cell induction, for example, DMEM, DMEM / F12 or DME culture solution containing 10-15% FBS (these culture solutions include LIF, penicillin / streptomycin, puromycin, L-glutamine). , Non-essential amino acids, β-mercaptoethanol, etc.) or a commercially available culture medium [eg, culture medium for mouse ES cell culture (TX-WES culture medium, THROMBO X), primate ES cell culture Medium (primate ES / iPS cell culture medium, ReproCELL), serum-free medium (mTeSR, Stemcell Technology)] and the like.

As an example of the culture method, for example, in the presence of 5% CO ₂ at 37 ° C., the somatic cell and the reprogramming factor are brought into contact with DMEM or DMEM / F12 containing 10% FBS for about 4 to 7 days. Then, re-spread the cells on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.), and use bFGF-containing primate ES cell culture medium about 10 days after contact between the somatic cells and the reprogramming factor. Culturing and generating iPS-like colonies about 30 to about 45 days or more after the contact.

Alternatively, 10% FBS-containing DMEM medium (including LIF, penicillin / streptomycin, etc.) on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.) in the presence of 5% CO ₂ at 37 ° C. Can be suitably included with puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.) and can generate ES-like colonies after about 25 to about 30 days or more . Desirably, instead of feeder cells, somatic cells to be reprogrammed themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or extracellular matrix (eg, Laminin- 5 (WO2009 / 123349) and Matrigel (BD)) are exemplified.

  In addition, a method of culturing using a medium not containing serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci USA 106.15720-15725). Furthermore, in order to increase establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845).

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

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

(E) ES cells derived from cloned embryos obtained by nuclear transfer (ntES cells)
An ntES cell is an ES cell derived from a cloned embryo produced by a nuclear transfer technique and has almost the same characteristics as an ES cell derived from a fertilized egg (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. (2007), Nature, 450: 497-502). That is, an ES cell established from an inner cell mass of a blastocyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with a somatic cell nucleus is an ntES (nuclear transfer ES) cell. For the production of ntES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (above) is used (Kiyaka Wakayama). (2008), Experimental Medicine, 26, 5 (extra number), 47-52). Nuclear transfer can be initialized by injecting a somatic cell nucleus into a mammal's enucleated unfertilized egg and culturing for several hours.

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

  In the present invention, pluripotent stem cells into which a gene causing MND is introduced can be used in addition to iPS cells derived from somatic cells of MND patients from the viewpoint of producing MND pathological model cells. In the case of ALS, examples of causative genes include SOD1, C9ORF72, TDP-43, FUS, PRN1, EPH4N, ANG, UBQLN, and HNPNPA, and these mutant genes can be introduced into pluripotent stem cells. For example, mutant SOD1 (A4V, G37R, G41D, H46R, G85R, D90A, G93A, G93S, I112T, I113T, L114F or S134N mutations are exemplified), mutant TDP-43 (M337V, Q343R, A315T mutations are exemplified) And pluripotent stem cells obtained by introducing). In the case of SBMA, a pluripotent stem cell obtained by introducing a mutant androgen receptor (AR) gene with an abnormally prolonged CAG repeat, and in the case of SMA, an SMN1 gene in which the seventh and eighth exon regions are deleted. Can be mentioned.

2. Nucleic acid encoding motor neuron differentiation factor (Lhx3, Ngn2 and Isl1) In step (1) of the production method of the present invention, Lhx3, Ngn2 and Isl1 (hereinafter referred to as “motor neuron differentiation factor (MNation factor) of the present invention”) The nucleic acid encoding (sometimes collectively) is introduced into the pluripotent stem cells.
In the present invention, the nucleic acid encoding Lhx3 is not particularly limited as long as it is a nucleic acid encoding a protein known as LIM homeobox 3. For example, the accession number: NM_001039653 (mouse) or NM_014564 or NM_178138 (human) in the NCBI database ) Registered polynucleotides, transcriptional variants, splicing variants and orthologs thereof, and those complementary to the complementary strand sequences of these nucleic acids under a stringent condition. It may be.

  In the present invention, the nucleic acid encoding Ngn2 is not particularly limited as long as it is a nucleic acid encoding a protein known as Neurogenin 2. For example, the accession number: NM_009718 (mouse) or NM_024019 (human) in the NCBI database The polynucleotide has a complementary relationship to the extent that it can hybridize under stringent conditions to the polynucleotide, its transcriptional variant, splicing variant and their orthologs, and the complementary strand sequences of these nucleic acids. It's okay.

  In the present invention, the nucleic acid encoding Isl1 is not particularly limited as long as it is a nucleic acid encoding a protein known as Islet 1, and is not particularly limited as long as it is a nucleic acid encoding NCBI protein. No: NM_021459 (mouse), or polynucleotides registered as NM_002202 (human), its transcriptional variants, splicing variants and their orthologs, and hybridizes to the complementary strand sequences of these nucleic acids under stringent conditions It is possible to have a complementary relationship to the extent that it is possible.

  As used herein, “stringent conditions” refers to complexes or probes as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). Can be determined based on the melting temperature (Tm) of the nucleic acid binding. For example, as washing conditions after hybridization, the conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” can be mentioned. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although not particularly limited, washing is performed under more severe hybridization conditions such as “0.5 × SSC, 0.1% SDS, 42 ° C.”, more strictly “0.1 × SSC, 0.1% SDS, 65 ° C.” However, the conditions for maintaining the hybridized state between the positive strand and the complementary strand can be mentioned. Specifically, as such a complementary strand, a strand comprising a base sequence that is completely complementary to the target positive strand base sequence, and at least 90%, preferably 95% or more, more preferably, the strand. Can be exemplified by a chain composed of a base sequence having 97% or more, more preferably 98% or more, particularly preferably 99% or more.

The nucleic acid encoding the MNation factor (Lhx3, Ngn2, Isl1) may be DNA or RNA. Preferred is double-stranded DNA or single-stranded RNA. For example, in the case of cDNA serving as a template for reconstitution of Sendai virus vector, the nucleic acid encoding Lhx3, Ngn2, or Isl1 is in the form of double-stranded DNA and is actually introduced into pluripotent stem cells On vectors (including the form of viral particles containing envelope proteins as well as the RNP complex consisting of genomic RNA and NP, P, and L proteins), the nucleic acid encoding these Mn factors is a minus-strand RNA. A nucleic acid encoding Lhx3, Ngn2, or Isl1 that is in the form and individually transcribed from the vector in the introduced cell is in the form of a plus-strand RNA.

  More specifically, the nucleic acid encoding the MNation factor of the present invention includes mouse-derived Lhx3 cDNA (SEQ ID NO: 1) and Ngn2 cDNA inserted into the pAd / PL-DEST vector described in the Examples below. (SEQ ID NO: 3) and Isl1 cDNA (SEQ ID NO: 5). In the present invention, since a Sendai virus vector is used for introduction of the MNation factor, when the nucleic acid encoding the MNation factor is transcribed into minus-strand RNA, the transcription termination signal (E) sequence is the nucleic acid sequence. In some cases, it is necessary to introduce a mutation into a part of the nucleotides in the nucleic acid sequence so that the function of the MNation factor is not impaired. In addition, if there are 6 or more continuous A regions in the plus strand sequence, mutations are likely to occur at the corresponding sites on the SeV vector, so silent mutations are introduced into the regions and the continuous A is 5 or less. It is desirable to modify so that For example, the 30th A of the Isl1 cDNA represented by SEQ ID NO: 5 is substituted with G.

  In one embodiment, in addition to nucleic acids encoding Lhx3, Ngn2 and Isl1, any other, as long as it does not inhibit motor neuron induction by the three factors (preferably improves motor neuron induction efficiency by the three factors) Nucleic acids encoding proteinaceous factors may be introduced into pluripotent stem cells. Examples of such additional MNation factor include nucleic acids encoding Ascl1, Brn2, Myt11 and HB9.

3. Sendai virus vector Nucleic acids encoding the MNation factor (Lhx3, Ngn2 and Isl1) of the present invention are inserted together into a single Sendai virus vector.
In this specification, a viral vector means a vector that has a genomic nucleic acid derived from the virus and can express the gene by incorporating a transgene into the nucleic acid.
Sendai virus is one of the Mononegavirales viruses and belongs to the Paramyxoviridae family (including Paramyxovirus, Morbillivirus, Rubulavirus, and Pnemovirus genera) and encodes a single negative chain (virus protein). RNA of the sense strand (antisense strand) is included as a genome. Negative strand RNA is also called negative strand RNA. The Sendai virus vector is a non-chromosomal viral vector, and since the vector is expressed in the cytoplasm, there is no risk of the transgene being integrated into the host chromosome. Therefore, the safety is high, and the vector can be removed from the introduced cell after the purpose is achieved.

  The Sendai virus vector in the present invention is a complex composed of a virus core, a complex of a virus genome and a virus protein, or a non-infectious virus particle in addition to an infectious virus particle. Complexes with the ability to express the onboard gene are included. For example, a ribonucleoprotein (virus core) consisting of a Sendai virus genome and a Sendai virus protein (NP, P, and L protein) that binds to the genome can express the transgene in the cell when introduced into the cell. Yes (disclosed in WO 00/70055). The introduction into the cells may be appropriately performed using a transfection reagent or the like. Therefore, such ribonucleoprotein (RNP) is also included in the Sendai virus vector in the present invention.

  Sendai virus genome is NP (nucleocapsid) gene, P (phospho) gene, M (matrix) gene, F (fusion) gene, HN (hemagglutinin / neuraminidase) in order from 3 'end to 5' end ) Gene and L (large) gene. Among these, Sendai virus can sufficiently function as a vector if it has the NP gene, P gene, and L gene, and it replicates the genome in the cell, and in the present invention (in the present invention, Lhx3, Ngn2, and Isl -1) can be expressed. Since Sendai virus has minus-strand RNA in the genome, the 3 'side of the genome is upstream and the 5' side is downstream.

The accession number of the base sequence database of each of the above genes of Sendai virus is, for example, M29343, M30202, M30203, M30204, M51331, M55565, M69046, X17218 for the NP gene, and M30202, M30203, M30204, for the P gene. For M55565, M69046, X00583, X17007, X17008, M gene, D11446, K02742, M30202, M30203, M30204, M69046, U31956, X00584, X53056, and F gene, D00152, D11446, D17334, D17335, M30202, M30203, For M30204, M69046, X00152, X02131, HN genes, D26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808, X56131, and L genes, D00053, M30202, M30203, M30204, M69040, X00587, X58886 It can be specified by referring.
However, a plurality of strains are known for Sendai virus, and there are also genes having sequences other than those exemplified above due to differences in strains. Sendai virus vectors having viral genes derived from any of these genes are also useful as Sendai virus vectors in the present invention. For example, the Sendai virus vector in the present invention is 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the coding sequence of any of the above viral genes. Includes a nucleotide sequence that has sex. The Sendai virus vector in the present invention is, for example, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more with the amino acid sequence encoded by the coding sequence of any of the above viral genes. Or a nucleotide sequence encoding an amino acid sequence having 99% or more identity. The Sendai virus vector in the present invention is, for example, within 10 amino acids, preferably within 9, within 8, within 7, within 6 within the amino acid sequence encoded by any of the above viral gene coding sequences. An amino acid sequence in which no more than 5, no more than 4, no more than 3, no more than 2, or 1 amino acid is substituted, inserted, deleted, and / or added, and retains the function of each gene product It includes a base sequence encoding a polypeptide.

  The sequence referred to by the database accession number such as the base sequence and amino acid sequence described in this specification refers to the sequence as of the filing date of the present application, and is specified as the sequence as of the filing date of the present application. The sequence at each time point can be specified by referring to the revision history of the database.

  The Sendai virus vector used in the present embodiment may be a derivative, and examples of the derivative include a virus whose virus gene has been modified, a virus that has been chemically modified, and the like so as not to impair the gene transfer ability by the virus. included.

  Sendai virus may also be derived from natural strains, wild strains, mutant strains, laboratory passage strains, artificially constructed strains, and the like. An example is Z strain (disclosed in Medical Journal of Osaka University Vol.6, No.1, March 1955 p1-15). That is, as long as the target function can be achieved, the virus may be a virus vector having the same structure as a virus isolated from nature, or a virus artificially modified by genetic recombination. For example, any gene possessed by the wild-type virus may be mutated or defective. It is also possible to use incomplete viruses such as DI particles (disclosed in J. Virol. 68: 8413-8417, 1994). For example, a virus having a mutation or deletion in at least one gene encoding a viral envelope protein or outer shell protein can be preferably used. Such a viral vector is, for example, a viral vector that can replicate the genome in infected cells but cannot form infectious viral particles. Such a transmission ability-deficient virus vector is highly safe because there is no concern of spreading infection around it. For example, viral vectors that do not contain at least one gene encoding an envelope protein or spike protein such as F and / or HN, or a combination thereof can be used (WO 00/70055, WO 00/70070, Li , H.-O. et al., J. Virol. 74 (14) 6564-6569 (2000)). If proteins necessary for genome replication (for example, NP, P, and L proteins) are encoded in genomic RNA, the genome can be amplified in infected cells. In order to produce a defective virus, for example, a defective gene product or a protein capable of complementing it is supplied exogenously in virus-producing cells (WO 00/70055, WO 00/70070, Li, H.-O et al., J. Virol. 74 (14) 6564-6569 (2000)). Further, when a viral vector is recovered as an RNP (for example, an RNP composed of N, L, P protein, and genomic RNA), the vector can be produced without complementing the envelope protein.

Suitable Sendai virus vectors in the present invention include, for example, mutations of G69E, T116A, and A183S in the M protein, mutations of A262T, G264, and K461G in the HN protein, L511F mutation in the P protein, and N1197S in the L protein. An F gene deletion-type Sendai virus vector (for example, Z strain) having a K1795E mutation may be used, and a vector in which a mutation of TS 7, TS 12, TS 13, TS 14, or TS 15 is further introduced into this vector is more preferable. . Specifically, SeV18 + / TSΔF (WO 2010/008054, WO 2003/025570), SeV (PM) / TSΔF, and further, mutations in TS 7, TS 12, TS 13, TS 14, or TS 15 Examples thereof include, but are not limited to, introduced vectors.
“TSΔF” has mutations of G69E, T116A, and A183S in the M protein, mutations of A262T, G264, and K461G in the HN protein, L511F mutation in the P protein, and N1197S and K1795E mutations in the L protein. To be deleted.

The position at which the nucleic acid encoding Lhx3 (L), the nucleic acid encoding Ngn2 (N) and the nucleic acid encoding Isl-1 (I) is not particularly limited, but L, N and 3 ′ to 5 ′ It is preferable to arrange in the order of I. These nucleic acids may be adjacent to each other (LNI), or any of the Sendai virus genomic genes may be interposed between them. That is, only one of L, N and I is upstream of the NP gene, between the NP gene and the P gene, between the P gene and the M gene, between the M gene and the HN gene, or between the HN gene and the L gene. It may be inserted between genes, or two or more (LN, NI, or LNI) may be inserted at any of the above positions. Preferably, L, N and I are integrated in this order immediately after the P gene, ie immediately downstream of the P gene (immediately 5 'to the minus-strand RNA genome). In this case, another transcription unit (for example, a transcription unit encoding a gene encoding a protein) is not included between the P gene and L. When L, N, and I are arranged in this order on the Sendai virus genome, L is arranged on the 3 ′ most side among the three nucleic acids, and I is arranged on the 5 ′ side most.
As described above, in a preferred embodiment, L, N, and I can be inserted between the P gene and the M gene, but even when inserted at other locations, the overall expression level changes. However, although there is a possibility that the induction efficiency of the motor neuron is different, the differentiation induction itself to the target motor neuron is possible in the same way.

  Each MNation factor is preferably sandwiched between a transcription initiation (S) sequence and a transcription termination (E) sequence. A region sandwiched between the S sequence and the E sequence becomes one transcription unit. An intervening (I) sequence serving as a spacer can be appropriately inserted between the E sequence of one gene and the S sequence of the next gene. For example, when L, N, and I are adjacent to each other in this order, a nucleic acid having the structure S- [L] -E-I-S- [N] -E-I-S- [I] -E is inserted into the Sendai virus genome.

In this embodiment, reconstitution of a recombinant Sendai virus vector having a MNation factor can be performed using a known method.
Specifically, (a) a cell that expresses a Sendai virus genomic RNA (minus strand) or its complementary strand (plus strand) and a viral protein (N, P, and L) necessary for virus particle formation. And (b) a step of recovering the culture supernatant containing the produced virus. Viral proteins necessary for particle formation may be expressed from transcribed viral genomic RNA, or may be supplied to trans from other than genomic RNA. For example, expression plasmids encoding N, P, and L proteins can be introduced into cells and supplied. If the genomic RNA lacks a viral gene necessary for particle formation, the virus gene can be separately expressed in virus-producing cells to complement particle formation. In order to express a viral protein or RNA genome in a cell, a vector in which DNA encoding the protein or genomic RNA is linked downstream of an appropriate promoter that functions in the host cell is introduced into the host cell. The transcribed genomic RNA is replicated in the presence of viral proteins to form infectious viral particles. When a defective virus lacking a gene such as an envelope protein is produced, the defective protein or another viral protein capable of complementing its function can be expressed in the virus-producing cell.

  In addition, Sendai virus can be produced using the following known methods (WO 97/16539; WO 97/16538; WO 00/70055; WO 00/70070; WO 01/18223; WO 03). WO 2005/071092; WO 2006/137517; WO 2007/083644; WO 2008/007581; Hasan, MK et al., J. Gen. Virol. 78: 2813-2820, 1997, Kato, A. et al ., 1997, EMBO J. 16: 578-587 and Yu, D. et al., 1997, Genes Cells 2: 457-466; Durbin, AP et al., 1997, Virology 235: 323-332; Whelan, SP et al., 1995, Proc. Natl. Acad. Sci. USA 92: 8388-8392; Schnell. MJ et al., 1994, EMBO J. 13: 4195-4203; Radecke, F. et al., 1995, EMBO J. 14: 5773-5784; Lawson, ND et al., Proc. Natl. Acad. Sci. USA 92: 4477-4481; Garcin, D. et al., 1995, EMBO J. 14: 6087-6094; , A. et al., 1996, Genes Cells 1: 569-579; Baron, MD and Barrett, T., 1997, J. Virol. 71: 1265-1271; Bridgen, A. and Elliott, RM, 1996, Proc Natl. Acad. Sci. USA 93: 15400-15404; Tokusumi, T. et al. Virus Res. 2002: 86; 33-38, Li, H.-O. et al., J. Virol. 2000: 74; 6564-6569).

4). Method for Introducing Nucleic Acid A Sendai virus vector carrying a nucleic acid encoding a MNation factor thus obtained is added to the medium of a pluripotent stem cell by adding the vector (Sendai virus particle) to infect the cell. Are introduced into the cells. The dose of the vector can be appropriately adjusted. For example, the multiplicity of infection (MOI) is 0.1 or more, preferably 0.3 or more, 0.5 or more, 1 or more, 2 or more, or 3 or more, and 100 or less, preferably 90 or less. 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, or 5 or less. Preferably, the infection is at a MOI of 0.3-100, more preferably a MOI of 0.5-50, a MOI of 1-40, a MOI of 1-30, a MOI of 2-30, or a MOI of 3-30.
Alternatively, when the Sendai virus vector is in the form of RNP, it can be introduced into pluripotent stem cells by techniques such as electroporation, lipofection, and microinjection.

  In the production method of the present invention, introduction into a Sendai virus vector containing a nucleic acid encoding a MNation factor into a pluripotent stem cell is preferably performed simultaneously with changing the pluripotent stem cell to differentiation-inducing conditions. In addition, since the expression period of the introduced MNation factor does not suffer from disadvantages in the production of motoneurons because it becomes long, no particular upper limit is set, but at least 2 days, preferably 3 days or more, It is preferably maintained for 4 days or more, 5 days or more, 6 days or more, or 7 days or more. More preferably, it is 2 days or more and 14 days or less. In the present invention, by using a sustained-expression type Sendai virus vector, the above-mentioned MNation factor can be expressed continuously. After a desired period of time, for example, by introducing an siRNA specific to an arbitrary gene sequence of the Sendai virus genome, for example, a sequence in the L gene, the virus vector can be eliminated.

5. Step of culturing gene-introduced pluripotent stem cell In the present invention, the condition for inducing differentiation of a motor neuron is culturing in a basic medium to which a differentiation signal factor for the motor neuron is added. Here, the motoneuron differentiation signal factor is not particularly limited as long as it is one or more signal transduction factors that can promote differentiation induction into motoneurons by the MNation factor introduced in the above step, for example, retinoic acid, SHH signal stimulants and neurotrophic factors.
As the basic medium, for example, Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 Examples include a medium, Fischer's medium, Neurobasal Medium (Life Technologies), and a mixed medium thereof. The basic medium may contain serum or may be serum-free. If necessary, the medium may be, for example, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acid, May contain one or more serum substitutes such as insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids It may also contain one or more substances such as vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenate, progesterone and putrescine. A preferred basal medium is a mixed medium of DMEM and F12 containing insulin, apotransferrin, selenate, progesterone and putrescine. In the present invention, the culture is preferably performed in a medium in which retinoic acid, an SHH signal stimulator, and a neurotrophic factor are appropriately added to this basic medium.

  In the present invention, a SHH (Sonic hedgehog) signal stimulator is defined as a substance that causes the inhibition of Smoothened (Smo) caused by SHH binding to the receptor Patched (Ptch1) and the subsequent activation of Gli2. For example, SHH, Hh-Ag1.5 (Li, X., et al., Nature Biotechnology, 23, 215-221 (2005)), Smoothened Agonist, SAG (N-Methyl-N '-(3-pyridinylbenzyl ) -N '-(3-chlorobenzo [b] thiophene-2-carbonyl) -1,4-diaminocyclohexane), 20a-hydroxycholesterol, Purmorphamine and derivatives thereof (Stanton BZ, Peng LF, Mol Biosyst. 6: 44-54, 2010). Preferably, it may be SAG.

In the present invention, a neurotrophic factor is a ligand to a membrane receptor that plays an important role in the survival and function maintenance of motor neurons, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) , Neurotrophin 3 (NT-3), neurotrophin 4/5 (NT-4 / 5), neurotrophin 6 (NT-6), basic FGF, acidic FGF, FGF-5, epidermal growth factor ( EGF), hepatocyte growth factor (HGF), insulin, insulin-like growth factor 1 (IGF 1), insulin-like growth factor 2 (IGF2), glial cell-derived neurotrophic factor (GDNF), TGF-b2, TGF-b3, Examples include, but are not limited to, interleukin 6 (IL-6), ciliary neurotrophic factor (CNTF), and LIF.
A preferred neurotrophic factor in the present invention is one or more factors selected from GDNF, BDNF and NT-3.

  One feature of the production method of the present invention is that the culture step under the differentiation-inducing conditions can be carried out using a medium having a single composition without changing the composition of the differentiation signal factor. . As used herein, “substantially a single composition” means that other medium composition changes can be allowed unless the combination and / or concentration of differentiation signal factors to motor neurons is changed, For example, if a ROCK inhibitor (for example, Y-27632) is added to the medium on the day of nucleic acid introduction and the medium composition is not changed except for changing to a medium not containing the ROCK inhibitor after the next day, It can be said that it is a single composition.

The culture temperature in the differentiation induction step of the motor neuron of the present invention is not particularly limited, but is about 30 to 40 ° C., preferably about 37 ° C. The culture is performed in an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is , Preferably about 2-5%.

  The period of culturing the cells in the differentiation induction step after introduction of the nucleic acid encoding the MNation factor is not particularly limited as long as the production of motor neurons is confirmed, but for example, 2 days or more, preferably 3, 4, 5, 6 , 7, 10 or 14 days or longer.

  As used herein, the terms “inducing motor neurons” or “producing motor neurons” are used interchangeably to mean obtaining a population of cells containing motor neurons. In the present invention, “motor neuron” is defined as a cell expressing a motor neuron-specific marker gene such as HB9 and / or ChAT. Further, the cell population containing motor neurons obtained by the production method of the present invention does not express markers specific to motor neurons, but also includes neurons other than motor neurons that express neurons specific markers. Can be. In the present invention, “neuron” is defined as a cell expressing a neuron marker such as Tuj1 and / or MAP2.

  Whether or not motoneurons and / or neurons are induced by the production method of the present invention is, for example, related to the cell population obtained through the differentiation induction step, immunocytochemistry or quantitative PCR based on the aforementioned expression markers (qPCR) can be performed by observing the protein expression and / or mRNA expression by a technique known to those skilled in the art.

  According to the production method of the present invention, since nucleic acids encoding all MNation factors are introduced into a single Sendai virus vector and introduced, at least between the types of MNation factors introduced into pluripotent stem cells. There is no variation. Therefore, the production method of the present invention can provide a more homogeneous motor neuron population. This is related to homogeneity among motor neurons, but if the type of Mn factor to be introduced varies among cells, the probability that neurons can be differentiated but cannot be differentiated specifically into motor neurons. Is also expected to increase. When nucleic acids that actually encode Lhx3 and Ngn2 (and Isl1) are introduced on different Sendai virus vectors, the proportion of motor neurons in the total neurons in the resulting cell population is low. In contrast, according to the production method of the present invention in which all MNation factors are mounted on a single vector, the ratio of motor neurons in the whole neurons is, for example, 60% or more, preferably 70% or more, and more preferably 80%. Remarkably high at more than%. This strongly suggests that there is little variation among cells among motor neurons, and that more uniform motor neurons are obtained.

  Exercise induced from iPS cells derived from motor neuron disease (MND) patients and pluripotent stem cells (PSC) derived from model animals (such as mice) into which a mutant gene causing MND has been introduced by the production method of the present invention Neurons can well reproduce the disease phenotype in the patient or model animal from which they originated. For example, in the case of a pluripotent stem cell-derived motor neuron (ALS PSC-MN) having a mutated gene causing ALS, it exhibits a phenotype such as a decrease in cell survival rate or suppression of neurite outgrowth. Further, in the case of ALS PSC-MN having a mutation of SOD1, misfolding of SOD1 protein and aggregation of misfolded protein in motor neurons can be mentioned as ALS phenotypes. Alternatively, in the case of ALS PSC-MN having a mutation of TDP-43, an increase in the amount of TDP-43 protein in motor neurons and aggregation of insoluble TDP-43 in the cytoplasm can be mentioned as the ALS phenotype. In the case of SBMA PSC-MN having a mutated androgen receptor (AR) gene that causes SBMA, testosterone-induced AR aggregation can be cited as an SBMA phenotype. In the case of SMA PSC-MN with SMN1 mutations that cause SMA, SMA phenotypes include a decrease in the number of spinal motor neurons, suppression of neurite outgrowth, and apoptosis with caspase-3 and -8 activation. .

  In the present invention, a cell culture having a neuromuscular junction where myotube cells and motor neurons are adhered can be obtained by mixing myotube cells in the step of producing motor neurons from pluripotent stem cells. . The neuromuscular junction means a structure in which acetylcholine is released from the projection end of a nerve cell and can be received by a receptor present in a myotube cell. The neuromuscular junction can be confirmed, for example, by colocalization of SV2 in motor neurons and acetylcholine receptors in myotubes. Cell cultures containing neuromuscular junctions are useful in reproducing the pathologies caused by neuromuscular junction malformations (eg, myasthenia gravis and Lambert-Eaton myasthenia).

<Screening method of therapeutic agent for motor neuron disease>
The present invention is also derived from a motor neuron population derived from a motor neuron disease (MND) patient-derived iPS cell obtained by the production method of the present invention, or a pluripotent stem cell into which an MND causative gene has been introduced. Provided is a method for screening a candidate drug for MND using a population of motor neurons. The method is
(1) contacting the test substance with the motor neuron population;
(2) measuring the degree of motor neuron disease phenotype in the cell population; and
(3) including a step of selecting a test substance having an improved degree of the phenotype as a therapeutic drug candidate substance for motor neuron disease as compared with a case where the test substance is not contacted.

  As the phenotype measured in step (2), one or more phenotypes specific to the various MNDs described above can be appropriately selected. For example, in the case of ALS PSC-MN, the number of motor neurons, neurite length, SOD1 protein misfolding, cytosolic aggregation of insoluble TDP-43 protein, and the like can be used as indices.

  The method for measuring the number of cells is not particularly limited, but may be performed by measuring the number of living cells, or may be calculated by the reciprocal of the number of dead cells. The number of dead cells can be measured by measuring the absorbance using the MTT method, WST-1 method, WST-8 method, or TO (thiazole orange), PI (propidium iodide), 7AAD, calcein AM, or ethidium. Examples thereof include a method of staining with homodimer 1 (EthD-1) and counting using a flow cytometer, or a method of measuring LDH activity.

  The neurite length can be measured by visual observation, and for example, it may be measured using a cell image analyzer (in-cell analyzer). When using a cell image analyzer, a vector that expresses a fluorescent substance (for example, GFP) downstream of the promoter of HB9 can be incorporated into cells so that motor neurons can be specifically recognized.

  Detection of misfolded SOD1 protein and insoluble TDP-43 can be carried out by the methods described in Examples described later, respectively.

  Examples of test substances include cell extracts, cell culture supernatants, microbial fermentation products, marine organism extracts, plant extracts, purified proteins or crude proteins, peptides, non-peptide compounds, synthetic low molecular compounds, and Natural compounds are mentioned.

  The test substances are also (1) biological library, (2) synthetic library method using deconvolution, (3) “one-bead one-compound” library method, and (4) can be obtained using any of a number of approaches in combinatorial library methods known in the art, including synthetic library methods using affinity chromatography sorting. Biological library methods using affinity chromatography sorting are limited to peptide libraries, but other approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994) J. Med. Chem 37: 1233-51). Compound libraries can be found in solutions (see Houghten (1992) Bio / Techniques 13: 412-21) or beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555- 6) Bacteria (US Pat. No. 5,223,409), Spores (US Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-9) or phage (Scott and Smith (1990) Science 249: 386-90; Devlin (1990) Science 249: 404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87 : 6378-82; Felici (1991) J. Mol. Biol. 222: 301-10; US Patent Application Publication No. 2002/0103360).

  Compared to the case where the test substance was not contacted, the test substance that increased the number of motor neuron cells, extended the neurite, decreased the amount of misfolded SOD1 and insoluble TDP-43, Can be selected as a candidate substance for ALS treatment.

  Similarly, in the case of MND PSC-MN other than ALS, each disease-specific phenotype is detected by a method known per se, and the degree is compared between when the test substance is contacted and when it is not contacted. This makes it possible to select a therapeutic drug candidate substance for the MND.

  Although described with emphasis on its use as a drug candidate compound search system (medicine efficacy screening system), the MND PSC-MN of the present invention can be used for individual patients (or the same causative gene mutation) of an existing drug compound for the disease. It can also be used for sensitivity assessment (classification of patient groups (eg, responders and non-responders)). Furthermore, the present invention can be applied to identify drug sensitivity marker genes by comparing the results of comprehensive gene expression analysis among classified patient groups.

<Transplantation therapy using motoneurons derived from pluripotent stem cells>
On the other hand, motor neurons derived from pluripotent stem cells derived from healthy individuals can be used as transplantation therapeutic agents for neurodegenerative diseases and nerve damage including MND (for example, ALS, SBMA, SMA, etc.). Since PSC-MN obtained by the production method of the present invention does not incorporate MNation factors or vector elements into the chromosome, it can be used safely with low risk of tumorigenesis. Motor neurons are produced as parenteral preparations such as injections, suspensions, drops, etc. by mixing with pharmaceutically acceptable carriers according to conventional means. 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 preparation 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 a motor neuron is formulated as an aqueous suspension, the motor neuron may be suspended in the aqueous solution so as to be about 1 × 10 ⁵ to 1 × 10 ⁸ cells / mL. Motor neuron transplantation can be performed by injecting the suspension into a lesion of neurodegeneration or nerve injury. The number of cells to be administered can be appropriately changed depending on the degree of lesion, etc. For example, in the case of a human ALS patient, about 1 × 10 ⁵ to 1 × 10 ⁸ cells can be administered.

  The transplantation and drug therapy can be used in combination. As a concomitant drug, for example, when the target disease is ALS, riluzole (trade name: Riltech (registered trademark) (Sanofi)), which is an existing ALS treatment drug, or 1,3-diphenyl described in WO2012 / 029994 Urea derivatives or multikinase inhibitors, HMG-CoA reductase inhibitors described in WO2011 / 074690, or anacadic acid (Egawa, N et al, Sci Transl Med. 4 (145): 145ra104. Doi: 10.1126) Can be mentioned. When the target disease is SBMA, luteinizing hormone analogs such as leuprorelin acetate can be mentioned. These agents can be used, for example, in dosages / administration routes usually used for treatment (clinical trial) of ALS and SBMA.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

<Materials and methods>
1. Induction of human fibroblasts and preparation of iPS cells Human fibroblasts were obtained with written consent. iPS cells were prepared by the method described in Egawa N. et al., Sci Transl Med. 2012 Aug 1; 4 (145): 145ra104. After selecting iPS cell colonies, iPS cells were cultured and subcultured on the SNL feeder layer. As the medium, primate embryonic stem cell medium (ReproCELL) containing basic fibroblast growth factor (4 ng / ml; Wako Pure Chemical Industries) and 50 mg / ml penicillin and streptomycin was used. The medium was changed daily and iPS cells were passaged about once a week.

2. Induction of mouse embryonic fibroblasts and preparation of iPS cells Mouse embryonic fibroblasts (MEF) were prepared by the method described in Gurney ME. Et al., Science. 1994 Jun 17; 264 (5166): 1772-5. Were obtained from ALS model mice having SOD1 (G93A) mutation and their littermate control mice. The four reprogramming factors (Oct3 / 4, Sox2, Klf4 and c-Myc) are Okita K. et al., Nature. 2007 Jul 19; 448 (7151): 313-7 and Takahashi K. et al., Nat. Protoc. 2007; 2 (12): 3081-9 was introduced into MEF using a retroviral vector. Mouse iPS cells were cultured on SNL feeder cells.

3. Genotyping The human SOD1 gene was amplified from genomic DNA by PCR and directly sequenced using a 3100 Genetic Analyzer (Applied Biosystems, Life Technologies, Carlsbad, CA).

4. Construction of Sendai virus vector (production of SeV18 + Lhx3 / TS7ΔF)
1) Construction of a plasmid (pSeV18 + Lhx3 / TS7ΔF) for producing a SeV vector carrying the Lhx3 gene
Using the Lhx3 gene (SEQ ID NO: 1) mounted on plasmid DNA (pAd / PL-DEST-mLhx3) as a template, the 492th guanine (G) of the Lhx3 gene is adenine (A) by the Mutagenesis method using PCR. ) To eliminate the NotI recognition site. MLhx3_G492A_F (5′-CAGCCAAGCGaCCGCGCACCACC-3 ′ (SEQ ID NO: 7)) and mLhx3_G492A_R (5′-GGTGGTGCGCGGtCGCTTGGCTG-3 ′ (SEQ ID NO: 8)) were used as primers for introducing the 492nd mutation. Using the Lhx3 gene into which this mutation was introduced as a template, using Not1_mLhx3_EIS_N (5'-taagcggccgccaaggttcaATGGAAGCTCGCGGGG-3 '(SEQ ID NO: 9)) and mLhx3_EIS_Not1_C (5'-ttagcggccgcgatgaactttGTCTCACTacacGG Using KOD-Plus-Ver.2, 94 ° C for 2 minutes, (98 ° C for 10 seconds, 55 ° C for 30 seconds, 68 ° C for 1.5 minutes) for 30 cycles, 68 ° C for 5 minutes, 4 ° C for ∞ PCR was performed, and the amplified fragment was purified with a QIAquick PCR purification kit. The purified fragment was digested with Not I and separated by agarose gel electrophoresis, and then a band of about 1.3 kbp was excised and purified with QIAquick Gel Extraction kit. On the other hand, the pSeV18 + / TS7ΔF plasmid having a transgene insertion site (NotI site) upstream of the NP gene was digested with NotI and purified with the QIAquick PCR purification kit, then treated with alkaline phosphatase, and again with the QIAquick PCR purification kit. Purified. Next, the purified Lhx3 fragment was ligated to the NotI site of the pSeV18 + / TS7ΔF plasmid, cloned into Escherichia coli, cloned, and a clone with the correct base sequence was selected by sequencing to obtain the pSeV18 + Lhx3 / TS7ΔF plasmid.

2) Construction of plasmid (pSeV18 + Ngn2 / TS7ΔF) for preparation of SeV vector carrying Ngn2 gene
Using the Ngn2 gene (SEQ ID NO: 3) mounted on plasmid DNA (pAd / PL-DEST-mNgn2) as a template, Not1_mNeurog2_EIS_N (5'-taagcggccgccaaggttcaCTTATGTTCGTCAAATCTG-3 '(SEQ ID NO: 11)) and mNeurog2_EIS_notc_ttcct (ctcctgctctctctctctctctctctctct -3 '(SEQ ID NO: 12)) using KOD-Plus-Ver.2 and a cycle of 94 ° C for 2 minutes (98 ° C for 10 seconds, 55 ° C for 30 seconds, 68 ° C for 1.5 minutes) PCR was performed under the conditions of 30 cycles, 68 ° C. for 5 minutes, 4 ° C. ∞, and the amplified fragment was purified with the QIAquick PCR purification kit. The purified fragment was digested with Not I and separated by agarose gel electrophoresis, and then a band of about 0.9 kbp was cut out and purified with a QIAquick Gel Extraction kit. On the other hand, the pSeV18 + / TS7ΔF plasmid having a transgene insertion site (NotI site) upstream of the NP gene was digested with NotI and purified with the QIAquick PCR purification kit, then treated with alkaline phosphatase, and again with the QIAquick PCR purification kit. Purified. Next, the purified Ngn2 fragment was ligated to the NotI site of the pSeV18 + / TS7ΔF plasmid, cloned into Escherichia coli, cloned, and a clone with the correct base sequence was selected by sequencing to obtain the pSeV18 + Ngn2 / TS7ΔF plasmid.

3) Construction of plasmid (pSeV18 + Isl1 / TS7ΔF) for preparing SeV vector carrying Isl1 gene
Using the Isl1 gene (SEQ ID NO: 5) mounted on plasmid DNA (pAd / PL-DEST-mIsl1) as a template, among the adenine (A) in which the Isl1 gene continues, the 30th adenine is obtained by the Mutagenesis method using PCR. (A) was converted to guanine (G), reducing the adenine (A) sequence. In addition, the 513th guanine (G) was converted to adenine (A) to eliminate the NotI recognition site. Using the ISL1_A30G_F (5'-GATCCACCAAAAAAgAAACGTCTGATTTCC-3 '(SEQ ID NO: 13)) and ISL1_A30G_R (5'-GGAAATCAGACGTTTcTTTTTTGGTGGATC-3' (SEQ ID NO: 14)) as primers for the 30th mutagenesis, the 513 th mutagenesis MIsl1_G513G_F (5′-GCCAGCTCTGCGaCCGCACGTCCAC-3 ′ (SEQ ID NO: 15)) and mIsl1_G513G_R (5′-GTGGACGTGCGGtCGCAGAGCTGGC-3 ′ (SEQ ID NO: 16)) were used as primers. Notl_mIsl1_EIS_N (5'-taagcggccgccaaggttcaCTTATGGGAGACATGGGC-3 '(SEQ ID NO: 17)) and ISL1_EIS_Not1_C (5'-aatgcggccgcgctactctcctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctactctcctctctctctctctctctctctctctctctctctctctctctctctctctctgctgctgctgcgctgctgcgcgcgctgcgctgcgcgctgcgcgcgcgcgggggggggggggggg3gg) , Using KOD-Plus-Ver.2, 30 cycles of 94 ℃ 2 minutes, (98 ℃ 10 seconds, 55 ℃ 30 seconds, 68 ℃ 1.5 minutes), 68 ℃ 5 minutes, 4 ℃ ∞ PCR was performed under the conditions described above, and the amplified fragment was purified with a QIAquick PCR purification kit. The purified fragment was digested with Not I and separated by agarose gel electrophoresis, and then a band of about 1.1 kbp was excised and purified with a QIAquick Gel Extraction kit. On the other hand, the pSeV18 + / TS7ΔF plasmid having a transgene insertion site (NotI site) upstream of the NP gene was digested with NotI and purified with the QIAquick PCR purification kit, then treated with alkaline phosphatase, and again with the QIAquick PCR purification kit. Purified. Next, the purified Isl1 fragment was ligated to the NotI site of the pSeV18 + / TS7ΔF plasmid, cloned into Escherichia coli, cloned, and a clone with the correct base sequence was selected by sequencing to obtain the pSeV18 + Isl1 / TS7ΔF plasmid.

4) Construction of a plasmid (pSeV (PM) Lhx3-Ngn2-Isl1 / TSΔF) for the construction of a SeV vector carrying 3 genes of Lhx3-Ngn2-Isl1
The Lhx3 gene fragment was constructed by PCR using the Lhx3 gene loaded on the SeV18 + Lhx3 / TS7ΔF cDNA as a template. Using Not. Then, PCR was performed at 94 ° C for 2 minutes (98 ° C for 10 seconds, 55 ° C for 30 seconds, 68 ° C for 1.5 minutes) for 30 cycles, 68 ° C for 5 minutes, 4 ° C ∞, and the amplified Lhx3 gene fragment ( About 1.3 kbp) was purified with a QIAquick PCR purification kit.
The Ngn2 gene fragment was constructed by PCR using the Ngn2 gene loaded on the SeV18 + Ngn2 / TS7ΔF cDNA as a template. mLhx3-mNgn2_N (5'-gtaagaaaaacttagggtgaaagttcatccacctaacagccgccATGTTCGTCAAATCTG-3 '(SEQ ID NO: 20)) and mNgn2-mIsl1_C (5'-ggtgaaatctttcaccctaagtttttcttattctacggC21 Primer-Ver. Lhx3 gene amplified by PCR under the conditions of 94 ° C for 2 minutes, 30 cycles of 98 ° C for 10 seconds, 55 ° C for 30 seconds, 68 ° C for 1.5 minutes, 68 ° C for 5 minutes, 4 ° C ∞ The fragment (about 0.9 kbp) was purified with QIAquick PCR purification kit.
The construction of the Isl1 gene fragment was performed by PCR using the Isl1 gene mounted on the SeV18 + Isl1 / TS7ΔF cDNA as a template. Using mNgn2-mIsl1_N (5′-gaaagatttcacctaacacgccgccATGGGAGACATGGGCG-3 ′ (SEQ ID NO: 22)) and EIS-NotI-R (5′-acctgcggccgcgaactttcaccctaagtttttc-3 ′ (SEQ ID NO: 23)) as primers, KOD-Plus-Ver .2 was used to amplify by performing PCR at 94 ° C for 2 minutes (98 ° C for 10 seconds, 55 ° C for 30 seconds, 68 ° C for 1.5 minutes) under 30 cycles, 68 ° C for 5 minutes, and 4 ° C ∞. The Isl1 gene fragment (about 1.1 kbp) was purified with the QIAquick PCR purification kit.
Next, the Lhx3-Ngn2-Isl1 gene fragment was constructed by PCR using a mixture of the Lhx3 gene fragment, Ngn2 gene fragment and Isl1 gene fragment prepared by the aforementioned PCR as a template. Using Not1_mLhx3_EIS_N and EIS-NotI-R as primers, using KOD-Plus-Ver.2, 30 cycles of 94 ° C for 2 minutes (98 ° C for 10 seconds, 55 ° C for 30 seconds, 68 ° C for 1.5 minutes) PCR was performed under the conditions of 68 ° C. for 5 minutes and 4 ° C. ∞, and the amplified Lhx3-Ngn2-Isl1 gene fragment (about 3.2 kbp) was purified with the QIAquick PCR purification kit. Next, the purified Lhx3-Ngn2-Isl1 fragment was ligated to the NotI site of the pSeV (PM) / TSΔF plasmid, cloned into E. coli and cloned, and a clone with the correct base sequence was selected by sequencing, and pSeV (PM ) The Lhx3-Ngn2-Isl1 / TSΔF plasmid was obtained.

5) Construction of a plasmid (pSeV18 + EGFP / TS7ΔF) for preparing an EGFP gene-loaded SeV vector
Using the EGFP gene mounted on plasmid DNA (available from pCXLE-EGFP; addgene) as a template, Not1_EGFP_EIS_N2.seq (5'-attgcggccgccaaggttcacttATGGTGAGCAAGGGCGAGGAGCTGTTCACCGCTCTCGTCctcctctcctcctctcctctcctctcctctcctcctctcct -3 '(SEQ ID NO: 25)) using KOD-Plus-Ver.2 and a cycle of 94 ° C for 2 minutes (98 ° C for 10 seconds, 55 ° C for 30 seconds, 68 ° C for 1.5 minutes) PCR was performed under the conditions of 30 cycles, 68 ° C. for 5 minutes, 4 ° C. ∞, and the amplified fragment was purified with the QIAquick PCR purification kit. The purified fragment was digested with Not I and separated by agarose gel electrophoresis, and then a band of about 0.8 kbp was cut out and purified with a QIAquick Gel Extraction kit. On the other hand, the pSeV18 + / TS7ΔF plasmid having a transgene insertion site (NotI site) upstream of the NP gene was digested with NotI and purified with the QIAquick PCR purification kit, then treated with alkaline phosphatase, and again with the QIAquick PCR purification kit. Purified. Next, the purified EGFP fragment was ligated to the NotI site of the pSeV18 + / TS7ΔF plasmid, cloned into E. coli after cloning, and a clone with the correct base sequence was selected by sequencing to obtain the pSeV18 + EGFP / TS7ΔF plasmid.

6) Lhx3 gene loaded SeV vector (SeV18 + Lhx3 / TS7ΔF), Ngn2 gene loaded SeV vector (SeV18 + Ngn2 / TS7ΔF), Isl1 gene loaded SeV vector (SeV18 + Isl1 / TS7ΔF), Lhx3-Ngn2-Isl1 loaded 3 genes Construction (reconstruction) of SeV vector (SeV (PM) Lhx3-Ngn2-Isl1 / TSΔF) and EGFP gene-loaded SeV vector (SeV18 + EGFP / TS7ΔF)
The day before transfection, 6 6-well plates were seeded with 10 ⁶ cells of 293T / 17 cells per well and cultured in a 37 ° C. CO 2 incubator (under 5% CO 2 condition). PCAGGS-NP (0.5μg), pCAGGS-P4C (-) (0.5μg), pCAGGS-L (TDK) (2μg), pCAGGS-T7 (0.5μg), pCAGGS-F5R (0.5μg) (See WO2005 / 071085), as well as the above-mentioned plasmid for preparing the SeV vector loaded with Lhx3 gene (pSeV18 + Lhx3 / TS7ΔF), the plasmid for creating the SeV vector loaded with Ngn2 gene (pSeV18 + Ngn2 / TS7ΔF), and the SeV loaded with Isl1 gene Plasmid for vector construction (pSeV18 + Isl1 / TS7ΔF), plasmid for SeV vector construction (pSeV (PM) Lhx3-Ngn2-Isl1 / TSΔF) for Lhx3-Ngn2-Isl1 or SeV vector for EGFP gene Plasmid (pSeV18 + EGFP / TS7ΔF) (5.0 μg) was mixed, and transfection was performed using 15 μl of TransIT-LT1 (Mirus). The cells were cultured for 2 days in a 37 ° C. CO 2 incubator. Subsequently, cells expressing Sendai virus fusion protein (F protein) LLC-MK2 / F / A (Li, H.-O. et al., J. Virology 74. 6564-6569 (2000), WO00 / 70070) 293T / 17 cells transfected at a rate of 10 ⁶ cells per well and cultured in a CO 2 incubator at 32 ° C. for 1 day. The next day, the cell culture medium is removed, the cells are washed once with 1 ml of MEM medium (hereinafter PS / MEM) supplemented with penicillin streptomycin, and PS / MEM medium containing 2.5 μg / ml trypsin (hereinafter Try / PS / MEM). 1 ml / well was added and cultured in a CO 2 incubator at 32 ° C. for 2 days. Cultivation was continued while subcultured with LLC-MK2 / F / A cells in some cases while changing the medium every 3-4 days. A portion of the culture supernatant was checked for the presence or absence of vector recovery by hemagglutination analysis, and after a sufficient hemagglutination reaction was obtained, the culture supernatant was collected. RNA was collected from the collected culture supernatant using the QIAamp Viral RNA Mini Kit, and RT-PCR was performed targeting the loaded transcription factor region. The obtained RT-PCR product was confirmed to have the correct nucleotide sequence by sequencing, and the SeV18 + Lhx3 / TS7ΔF vector (hereinafter also referred to as SeV-L) and SeV18 + Ngn2 / TS7ΔF vector (hereinafter also referred to as SeV-N). ), SeV18 + Isl1 / TS7ΔF vector (hereinafter also referred to as SeV-I), eV (PM) Lhx3-Ngn2-Isl1 / TSΔF vector (hereinafter also referred to as SeV-LNI) and SeV18 + EGFP / TS7ΔF vector (hereinafter referred to as SeV) -Also called EGFP). Each Sendai virus solution obtained was rapidly frozen in liquid nitrogen and then stored at -80 ° C.

5. SeV MOI for transduction of ES / iPS cells
To determine the MOI of the SeV vector, SeV-EGFP was transduced into control iPS cells. iPS cells were treated with collagenase type IV, trypsin, and KSR (CTK) separation (ReproCELL) for 1 minute, separated into single cells with Accumax (Innovative Cell Technologies), and coated with Matrigel (Becton Dickinson) 96 Transfer to well plate. Cells were fixed on day 2 and day 4. Images were taken with In Cell Analyzer 6000 (GE Healthcare).

6. Differentiation of mouse iPS cells into motor neurons using SeV vectors
iPS cells are treated as a single cell by trypsinization, 0.5% N2 (Life Technologies), 1% B27 (Life Technologies), 1 μM retinoic acid (SIGMA), 1 μM smoothened agonist (Enzo Life Sciences), 10% ng / ml brain-derived neurotrophic factor (BDNF; R & D Systems), 10 ng / ml glial cell-derived neurotrophic factor (GDNF; R & D Systems), 10 ng / ml neurotrophin 3 (NT-3; R & D Systems) and 10 Coated with Matrigel containing MN medium containing 1: 1 mixture of Neurobasal medium (Life Technologies) supplemented with μM Y-27632 (Nacalai tesque) and DMEM / F12 (1: 1; 1 ×; Life Technologies) I sprinkled on the plate. At the same time, iPS cells were infected with SeV-LNI on day 0. The MOI was 5. The medium was changed to MN medium not containing Y-27632 on the 1st and 4th days. Cells were evaluated by immunocytochemistry on day 6.

7. Induction from human iPS cells to MN using SeV vector
iPS cells were treated with CTK separation solution for 2 minutes, and feeder cells were removed using PBS. Thereafter, iPS cells were separated into single cells using Accumax, and transferred onto a plate coated with Matrigel containing MN medium. At the same time, iPS cells were infected on day 0 with SeV-LNI or a combination of SeV-L, SeV-N, and SeV-I. The MOI was 10 in FIGS. 1 to 3, but when the MOI was 10, the SOD1-ALS iPS cell was dead, so the MOI was 5 in FIGS. The medium was changed to MN medium not containing Y-27632 on the first day, and thereafter changed every three days. In FIGS. 4 and 5, cells were treated with Accumax plus 10 μM Y-27632 and transferred on day 7 onto glass dishes coated with poly-L-lysine and Matrigel. Cells were evaluated on day 14 by immunocytochemistry and qPCR analysis.

8. RNA extraction, cDNA synthesis, and quantitative PCR (qPCR)
RNA was isolated using RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. cDNA was synthesized using the ReverTra Ace-α kit (Toyobo). qPCR was performed with StepOne Plus instrument (Applied Biosystems) using SYBR Premix Ex Taq II (Takara). Primer sequences are shown below (Table 1).

  In the table, SEQ ID NOs in the sequence listing of each primer are SEQ ID NOS: 26 to 33 in order from the top.

9. Co-culture of human motor neurons and human myoblasts
The Hu / E18 cell line was purchased from RIKEN BioResource Center. Hu5 / E18 cells were maintained and differentiated by the method reported by Hashimoto N. et al., Biochem Biophys Res Commun. 2006 Oct 6; 348 (4): 1383-8. Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Nacalai Tesque) with high glucose containing 20% fetal calf serum (Gibco). Cells were treated with 5 μg / ml holotransferrin bovine (SIGMA), 10 μg / ml insulin (bovine, SIGMA), 10 nM sodium selenite (SIGMA) and 2% 7 days prior to SeV-LNI infection of iPS cells. Differentiated into human myocytes in DMEM containing horse serum (Gibco). iPS cells were infected with SeV-LNI on day 0, separated with Accumax plus 10 μM Y-27632, and then transferred onto Hu5 / E18 culture plates on day 7. The medium was changed to MN medium. On day 14, cells were fixed in 4% paraformaldehyde (pH 7.4) for 30 minutes and evaluated by immunocytochemistry.

10. Electrophysiological recording Electrophysiological recording and analysis was performed on the 21st day with optical microscopy and differential interference (DIC) imaging using the method described in Egawa N. et al., Sci Transl Med. 2012 (supra). Conducted under combination. During electrophysiological recording, the cells are maintained at 30 ° C and from 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 26 mM NaHCO ₃ , 1.25 mM NaH ₂ PO ₄ , and 20 mM glucose Perfused continuously with an oxygenated Krebs-Ringer solution. Chloride composed of 140 mM KCl, 2 mM MgCl ₂ , 10 mM HEPES and 1 mM EGTA, adjusted to pH 7.4 using NaOH to examine whether iPS cell-derived motor neurons are functionally active The action potential was measured in current clamp mode using a potassium-based electrode solution. For recording, an EPC 9 amplifier (HEKA) was used, and the data was analyzed by Patchmaster software (HEKA). Primary astrocytes were obtained from P1 mice and cultured in DMEM containing 10% FBS.

11. Immunocytochemical cells were fixed with 4% paraformaldehyde (pH 7.4) for 30 minutes. Thereafter, the cells were permeabilized with 0.2% Triton X-100, and non-specific binding portions were blocked with Block Ace (snow mark). Cells were incubated with primary antibody at 4 ° C. overnight and with secondary antibody for 1 hour at room temperature. Fluorescence images were taken and motor neurons or neuronal percentage was calculated using an In Cell Analyzer 6000. In FIG. 5, the images were acquired using Delta Vision (GE Healthcare). The following primary antibodies were used: HB9 (DSHB, 1: 200), Tuj1 (COVANCE, 1: 2,000), Tuj1 (Chemicon, 1: 500), ChAT (Chemicon, 1: 100), misfolded SOD1 (MEDIMABS, B8H10, 1: 200), misfolded SOD1 (MEDIMABS, A5C3, 1: 200), TDP-43 (Proteintech, 1: 200), human Nanog (ReproCELL, 1: 500), SSEA4 (Millipore, 1: 200), And SSEA1 (Chemicon, 1: 1000).

12. Time-lapse imaging For time-lapse imaging, 35-mm glass bottom dishes (MatTek) were coated with poly-L-lysine (SIGMA) and Matrigel. Human iPS cells transduced with SeV-LNI were seeded on the dish on day 0. Medium was 0.5% N2 (Life Technologies), 1% B27 (Life Technologies), 1 μM retinoic acid (SIGMA), 1 μM smoothed agonist (Enzo Life Sciences), 10 ng / ml Replacement with FluoroBrite DMEM supplemented with trophic factor (BDNF; R & D Systems), 10 ng / ml glial cell-derived neurotrophic factor (GDNF; R & D Systems), and 10 ng / ml neurotrophin 3 (NT-3; R & D Systems) did. Time-lapse imaging was started 24 hours after plating, and BioStation IM-Q (Nikon) was used. Images were taken every 30 minutes.

13. Statistical analysis All data are presented as mean ± SEM. Data were analyzed by Student's t test or one-way analysis of variance, followed by Dunnette's post hoc comparison test. Statistical analysis was performed using SPSS version 2.1.

Example 1 Generation and Characterization of Human iPS Cells First, we report in Takahashi K. et al., Cell. 2007 (supra) and Egawa N. et al., Sci Transl Med. 2012 (supra). From the fibroblasts of familial ALS patients with mutant superoxide dismutase 1 (SOD1) by transducing the four transcription factors Oct3 / 4, Sox2, Klf4, and c-Myc using Human iPS cells were prepared. In addition, the outline | summary regarding the characteristic of several human iPS cell strains including the human iPS cell produced this time is shown below (Table 2).

  iPS cells were examined by immunocytochemistry against SSEA4 and NANOG, which are ES cell markers (FIG. 6a), and confirmed to retain the SOD1 gene mutation (FIG. 6b). Another family ALS, TAR DNA binding protein 43kDa (TDP-43) -mediated ALS (human TDP-43 ALS) patient-derived iPS cell line and control-derived iPS cell line were also generated by Egawa N. et al., ( The method described in the above) was used.

Example 2 Differentiation of Human iPS Cells into Motor Neurons by Three Separate SeV Vectors Human iPS cells were differentiated into motor neurons by the method described in FIG. In order to easily detect motor neurons, HB9-EGFP knock-in human iPS cells were used. On day 0, iPS cells were seeded on matrigel-coated dishes, and the medium was changed from ES cell medium to Neurobasal medium containing N2 supplement and B27 supplement. RA, smoothed agonist (SAG), and neurotrophic factor (NTF) were also added on day 0. For the induction of motor neurons, three separate vectors, SeV18 + Lhx3 / TS7ΔF (SeV-L, SEQ ID NO: 5), SeV18 + Ngn2 / TS7ΔF (SeV-N, SEQ ID NO: 6), and SeV18 + Isl-1 / TS7ΔF (SeV-I, SEQ ID NO: 7) was transduced into human iPS cells. To test the transduction efficiency of the SeV vector, SeV18 + EGFP / TS7ΔF (SeV-EGFP, SEQ ID NO: 9) was transferred to control iPS cells at 1, 3, 10, 30, and 100 multiplicity of infection (MOI). Was transduced with. An efficiency of 50% or more was observed when the MOI was 3, and the highest efficiency was observed when the MOI was 100. However, cell death increased with higher MOI. Therefore, an MOI of less than 30 was selected. Immunocytochemical analysis and quantitative PCR (qPCR) performed on day 14 revealed HB9-positive, ChAT-positive, and MAP2-positive neurons (FIGS. 1b and 1c).

Example 3 Transduction of all three transcription factors results in highest efficiency in generating HB9 and Tuj1 positive cells from human ES / iPS cells
Three vectors, SeV-L, SeV-N, and SeV-I, to determine what combination of Lhx3, Ngn2, and Isl-1 can most efficiently produce motor neurons from iPS cells 1-3 were transduced into human iPS cells, and the expression of Tuj1 and HB9 was evaluated by immunocytohistological techniques. When the combination of all three transcription factors was introduced and when the combination of SeV-L and SeV-N was introduced, neurons positive for both Tuj1 and HB9 were obtained, while SeV-N was introduced. In this case, Tuj1 positive and HB9 negative neurons were obtained (FIG. 2a). In the case of medium alone, neither neurons nor motor neurons were induced (0%). When all three factors were used for transduction, 20.6% ± 8.7% cells were neurons and 7.9% ± 1.9% cells were motor neurons. When only Lhx3 and Ngn2 were used, 21.2% ± 1.6% of cells were neurons and 3.9% ± 0.5% of cells were motor neurons (FIG. 2b). When all three factors were used, the motor neuron / neuron ratio was 43.9% ± 6.6%, and with Lhx3 and Ngn2, it was 18.2% ± 1.1%. The combination of all three factors produced motor neurons with the highest efficiency, but to a lesser extent, the combination of the two factors (Lhx3 and Ngn2) could produce motor neurons. This suggests that when all three transcription factors are transduced simultaneously, some of the motoneurons can be made by only two transcription factors. In addition, it was confirmed that motor neurons can be produced from human ES cells using this SeV vector through immunocytochemistry and qPCR analysis (FIG. 7).

Example 4 Induction and temporal imaging of motoneurons by Lhx3, Ngn2, and Isl-1 in a single SeV vector We have developed a single SeV vector encoding Lhx3, Ngn2, and Isl-1 ( SeV-LNI) was designed and the induction of motor neurons was examined (Fig. 3a). On day 14, both HB9 positive and ChAT positive neurons were observed, of which 6.4% ± 0.8% were neurons and 5.0% ± 0.9% were motor neurons (FIG. 3b). The ratio of motor neurons / neurons was 78.1% ± 6.7%. qPCR analysis showed that the expression levels of HB9, ChAT, and MAP2 were increased (FIG. 3c). The action potential of the generated motor neurons when co-cultured with primary astrocytes was observed by electrophysiological patch clamp method (FIG. 3d). Colocalization of HB9-EGFP positive neurites and acetylcholine receptor stained with α-bungarotoxin when motor neurons are co-cultured with human myocytes differentiated from human myoblast cell line Hu5 / E18 This confirmed the formation of the neuromuscular junction (FIG. 3e).

  In order to capture the time point when HB9 positive cells were generated, temporal imaging using HB9-EGFP knock-in iPS cells was performed. Temporal imaging of GFP started on day 1 and GFP positive cells were observed on day 2. The number of GFP positive cells gradually increased, but some disappeared over time. On day 3, a neuron-like morphology was observed (data not shown).

Example 5 SOD1-ALS induced by SeV-LNI Motor neurons show disease-specific phenotypes In order to confirm whether the method of the present invention can be applied to MND studies, we first started Takahashi K Using the methods reported in et al., Cell. 2007 (supra) and Egawa N. et al., Sci Transl Med. 2012 (supra), the four transcription factors Oct3 / 4, Sox2, Klf4, and c By transducing -Myc, mutant SOD1 (Gurney ME. Et al., Science. 1994 (described above)), mutant TDP-43 (Wegorzewska I. et al., Proc Natl Acad Sci US A. 2009 Nov 3; IPS cells were produced from ALS model mice having 106 (44): 18809-14.) And control mouse fetal fibroblasts littered with them (FIG. 6c-d). In addition, the outline | summary regarding the characteristic of several mouse | mouth iPS cell strains including the produced mouse | mouth iPS cell is shown below (Table 3).

  Next, the present inventors induced the produced iPS cells to motor neurons and examined their phenotype via immunocytochemistry (FIG. 4). Motor neurons from mouse SOD1-ALS iPS cells were positive for misfolded SOD1, whereas motor neurons from mouse control iPS cells were negative (FIG. 5a). Mouse TDP-43-ALS iPS cell-derived motoneurons did not show aggregation of TDP-43 on the cytoplasmic matrix (Fig. 5b), which is related to the histopathological findings of TDP-43 transgenic mice. This is consistent with the report (Hatzipetros T. et al., Brain Res. 2014 Oct 10; 1584: 59-72.). When human ALS iPS cells were differentiated into motoneurons using SeV-LNI, human SOD1-ALS and human TDP-43-ALS motoneurons aggregated misfolded SOD1 (Fig. 5c) and TDP-43, respectively. Aggregation on the cytoplasmic substrate (Figure 5d) was shown. From the above, it was shown that the motoneurons induced by the induction method of the present invention show a disease-specific phenotype in patients or model mice from which iPS cells originate.

Example 6 Induction of motoneurons from human ES cells by a single SeV vector SeV-LNI In the same manner as in Example 4, we performed a single SeV encoding Lhx3, Ngn2, and Isl-1. A vector (SeV-LNI) was used to test the induction of motor neurons from human ES cells (FIG. 6a). On day 14, both HB9 positive neurons and ChAT positive neurons were observed (FIG. 6b). qPCR analysis showed that the expression levels of HB9, ChAT, and MAP2 were increased (FIG. 6c).

  While the 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. Thus, it is intended that the present invention be implemented in ways other than those described in detail herein. That is, the present 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 made explicit.

  Motor neurons obtained using the induction method of the present invention have no fear that the transduced gene is integrated into the host cell genome, and the expression level of the introduced transcription factor is relatively uniform. Therefore, it is considered that the motoneurons derived from pluripotent stem cells derived from motor neuron disease patients including ALS or model animals thereof using the method of the present invention more accurately reflect the pathology of the disease. . Therefore, it is considered to be extremely useful in research for screening candidate substances for treatment or prevention of these diseases.

  This application is based on Japanese Patent Application No. 2016-112289 (application date: June 3, 2016) for which it applied in Japan, The content is altogether included in this specification.

Claims (17)

A method for producing motor neurons from pluripotent stem cells, comprising the following steps:
(1) introducing a single Sendai virus vector into a pluripotent stem cell comprising a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2 and a nucleic acid encoding Isl1, and
(2) A step of culturing the pluripotent stem cells for 2 days or more.   The method according to claim 1, wherein the Sendai virus vector comprises a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2, and a nucleic acid encoding Isl1 in this order from the 3 'side to the 5' side.   The method according to claim 1 or 2, wherein the culturing step is performed in a medium containing a single compositional motor neuron differentiation signal factor throughout the entire period.   4. The method of claim 3, wherein the motor neuron differentiation signal factor comprises retinoic acid, a sonic hedgehog (SHH) signal stimulator and a neurotrophic factor.   The method according to any one of claims 1 to 4, wherein the proportion of motor neurons in the total obtained neurons is 60% or more.   6. The method according to any one of claims 1 to 5, wherein the pluripotent stem cells are derived from an animal exhibiting a motor neuron disease phenotype.   The method of claim 6, wherein the motor neuron disease is amyotrophic lateral sclerosis (ALS).   The method according to any one of claims 1 to 7, wherein the pluripotent stem cell is an ES cell or an iPS cell.   The method according to any one of claims 1 to 8, wherein the pluripotent stem cell is derived from a human or a mouse.   The method according to claim 9, wherein the pluripotent stem cell is an iPS cell derived from an ALS patient.   A motor neuron population obtained by the method according to claim 1.   A motor neuron population that reproduces the phenotype of motor neuron disease obtained by the method according to any one of claims 6 to 10.   A Sendai virus vector comprising a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2 and a nucleic acid encoding Isl1.   14. The vector according to claim 13, comprising a nucleic acid encoding Lhx3, a nucleic acid encoding Ngn2 and a nucleic acid encoding Isl1 successively in this order from the 3 'side to the 5' side.   An agent for inducing motor neuron differentiation from pluripotent stem cells, comprising the vector according to claim 13 or 14. A screening method for a candidate drug for the treatment of motor neuron disease comprising the following steps:
(1) A step of bringing a test substance into contact with the motor neuron population according to claim 12,
(2) measuring the degree of motor neuron disease phenotype in the cell population; and
(3) A step of selecting a test substance having an improved degree of the phenotype as a therapeutic drug candidate substance for motor neuron disease as compared with a case where the test substance is not contacted.   The method according to claim 16, wherein the motor neuron disease is ALS.