Method for producing nerve cells

Abstract

The present application provides a method for producing, from pluripotent stem cells, neural progenitor cells of the midbrain floor plate region or dopaminergic neural progenitor cells, the method comprising (1) inducing differentiation of pluripotent stem cells into neural progenitor cells of the midbrain floor plate region, and optionally (2) inducing differentiation of the neural progenitor cells of the midbrain floor plate region, which are obtained by the differentiation induced in (1), into dopaminergic neural progenitor cells, wherein all or part of the differentiation induced in (1) includes culturing the cells in a medium containing an agonist for LPA receptor.

Description

Method for manufacturing nerve cells

[0001] This application relates to a method for producing nerve cells such as neural progenitor cells or dopamine neural progenitor cells in the midbrain floor plate region.

[0002] Methods for inducing nerve cells, such as dopamine progenitor cells, from pluripotent stem cells (ES cells and iPS cells, etc.) are being established (Non-patent documents 1-4). In particular, the use of dopamine progenitor cells differentiated from pluripotent stem cells is being clinically applied as a cell transplantation therapy for Parkinson's disease. Furthermore, human pluripotent stem cell-derived dopamine neurons and their progenitor cells, which can be manufactured in vitro, have high value for use in disease research and drug discovery.

[0003] In methods for differentiating pluripotent stem cells such as ES cells and iPS cells into neural progenitor cells, a challenge can arise because differentiation efficiency and stability depend on the state of the pluripotent stem cells and the differentiation medium (differentiation protocol). Therefore, there is a need for differentiation aids and improved differentiation methods that can stably achieve high differentiation efficiency.

[0004] In particular, when using culture media containing biologically derived raw materials, concerns arise not only regarding safety but also regarding the impact of lot-to-lot variations in the raw materials. Therefore, there is a need for differentiation induction methods that do not use compositions containing biologically derived raw materials such as KSR (Knockout Serum Replacement) or serum, and that provide stable differentiation induction efficiency.

[0005] Furthermore, it is known that the state of the pluripotent stem cells used as raw materials affects their responsiveness to differentiation induction protocols. For example, even with protocols that achieve induction efficiency of nearly 90% (number of dopamine neural progenitor cells / total number of cells), target cells may not be induced at all in certain pluripotent stem cell lines. Moreover, in autologous transplantation, since iPS cells are individually induced from the patient's own somatic cells, a more stable differentiation method is required because iPS cells with diverse properties are handled. For these reasons, there is a need for the development of technologies and protocols that can stably induce target cells such as dopamine neural progenitor cells without depending on the pluripotent stem cell line.

[0006] International Publication No. 2015 / 034012, International Publication No. 2017 / 183736

[0007] Kirkeby A, et al., Cell Rep. 2012; 1(6): 703-14.Doi D, et al., Stem Cell Reports. 2014; 2(3): 337-350.Kriks S, et al., Nature. 2011; 480(7378): 547-51.Kim T et al., Cell Stem Cell. 2021; 28(2): 343-355.Nolbrant S, et al., Nat Protocols. 2017; 12(9)1962-1979.Dottori M et al., Stem Cells. 2008; 26(5):1146-1154.

[0008] The object of this application is to provide a method for producing nerve cells such as neural progenitor cells or dopamine neural progenitor cells of the midbrain floor plate region, a culture medium that can be suitably used in said production method, a differentiation induction ability stabilizer for a culture medium for inducing differentiation from pluripotent stem cells to neural progenitor cells of the midbrain floor plate region, and a method for stabilizing the differentiation induction ability of a serum-free culture medium comprising chemically defined components including the differentiation induction agent from pluripotent stem cells to neural progenitor cells of the midbrain floor plate region.

[0009] The inventors, through diligent research, have discovered that using a culture medium containing lysophosphatidic acid (LPA) in the initial stages of differentiation induction from pluripotent stem cells to dopamine neural progenitor cells improves the efficiency of dopamine neural progenitor cell induction. Specifically, the inventors have found that by adding an agonist targeting the LPA receptor in the early stages of induction, in addition to the differentiation-inducing factors used in previously reported differentiation induction protocols from pluripotent stem cells to dopamine neural progenitor cells, they can increase the efficiency of induction into neural progenitor cells in the midbrain floor plate region, and as a result, successfully improve the efficiency of induction into dopamine neural progenitor cells.

[0010] In other words, the present inventors provide the following: [1] A method for producing neural progenitor cells of the mesencephalic floor plate region or dopaminergic neural progenitor cells from pluripotent stem cells, comprising (1) induction of differentiation from pluripotent stem cells to neural progenitor cells of the mesencephalic floor plate region, (2) which may also include induction of differentiation from the neural progenitor cells of the mesencephalic floor plate region obtained by the differentiation induction in (1) to dopaminergic neural progenitor cells, wherein in part or all of the differentiation induction in (1), the cells are cultured in a medium containing an agonist for the LPA receptor. [2] The method according to [1], wherein the medium used in the differentiation induction in (1) comprises one or more substances selected from one or more substances that inhibit SMAD signaling, a substance that activates Shh signaling, and a substance that activates Wnt signaling, and comprises one or more steps of culture. [3] The method according to [1] or [2], wherein the medium used in the differentiation induction in (1) does not contain serum. [4] The method for producing a product according to [3], wherein the differentiation induction in (1) is carried out in the absence of Knockout serum replacement (KSR). [5] The method for producing a product according to [3] or [4], wherein the culture medium used in the differentiation induction in (1) is a serum-free medium consisting of chemically defined components. [6] The method for producing a product according to any one of [1] to [5], wherein the LPA receptor agonist is an agonist for at least one of LPA1, LPA2, and LPA3. [7] The method for producing a product according to any one of [1] to [6], wherein the LPA receptor agonist is one or more substances selected from oleoyl-L-α-lysophosphatidic acid, (1-oleoyl LPA), 20:4 lysoPA, NAEPA, (2S)-OMPT, and VPC31143. [8] The method for producing the product according to any one of [1] to [7], wherein the differentiation induction in (1) comprises culturing in a medium which contains one or more substances that inhibit SMAD signals and which may also contain a substance that activates the Shh signal and / or a substance that activates the Wnt signal.[9] The method for producing the organism according to [8], wherein the differentiation induction in (1) comprises (i) culturing in a medium containing one or more substances that inhibit SMAD signals, substances that activate Shh signals, and substances that activate Wnt signals.

[10] The method for producing the organism according to [8] or [9], wherein the differentiation induction in (1) comprises culturing in a medium containing FGF8.

[11] The manufacturing method according to [8], wherein the differentiation induction in (1) comprises: (i) culturing in a medium which contains one or more substances that inhibit the SMAD signal and which may also contain a substance that activates the Shh signal and / or a substance that activates the Wnt signal; (ii) after obtaining cells in the culture in (i), culturing the cells in a medium which contains one or more substances that inhibit the SMAD signal and a substance that activates the Shh signal and which may also contain a substance that activates the Wnt signal; and (iii) after obtaining cells in the culture in (ii), culturing the cells in a medium which contains one or more substances that inhibit the SMAD signal, a substance that activates the Shh signal and a substance that activates the Wnt signal, and further (iv) after obtaining cells in the culture in (iii), culturing the cells in a medium which contains one or more substances that inhibit the SMAD signal and a substance that activates the Wnt signal.

[12] The method for producing cells according to

[11] , wherein the culture medium used for culturing (ii) and / or (iii) is further a medium containing FGF8.

[13] The method for producing cells according to [8], wherein the differentiation induction in (1) comprises: (I) culturing pluripotent stem cells in a medium containing one or more substances that inhibit SMAD signaling, a substance that activates Shh signaling, and a substance that activates Wnt signaling; (II) after obtaining cells in the culture of (I), culturing the cells in a medium containing one or more substances that inhibit SMAD signaling and a substance that activates Wnt signaling; and (III) after obtaining cells in the culture of (II), culturing the cells in a medium containing one or more substances that inhibit SMAD signaling.

[14] The method for producing cells according to any one of [1] to

[13] , further comprising culturing in a medium containing a ROCK inhibitor for any period within three days from the start of the differentiation induction in (1).

[15] The method for producing cells according to

[14] , comprising culturing in a medium containing the ROCK inhibitor, followed by culturing in a medium containing an agonist for the LPA receptor.

[16] The method for producing cells according to any one of [1] to

[15] , comprising inducing differentiation from neural progenitor cells of the mesencephalic floor plate region of (2) to dopaminergic neural progenitor cells, wherein the medium used in the differentiation induction of (2) is a medium containing neurotrophic factors and ascorbic acid or a derivative thereof.

[17] The method for producing cells according to

[16] , wherein the medium used in the differentiation induction of (2) further contains cAMP or a derivative thereof.

[18] The method for producing cells according to

[16] or

[17] , wherein the neurotrophic factors are one or more neurotrophic factors selected from the group consisting of BDNF, GDNF, NT-3, and TGF-β3.

[19] A serum-free medium comprising chemically defined components for inducing differentiation of pluripotent stem cells into neural progenitor cells of the mesencephalic floor plate region, the medium comprising an agonist for the LPA receptor.

[20] A differentiation-inducing agent for a medium for inducing differentiation of pluripotent stem cells into neural progenitor cells of the mesencephalic floor plate region, comprising an agonist for the LPA receptor.

[21] A method for stabilizing the differentiation-inducing ability of a serum-free medium comprising chemically defined components including a differentiation-inducing agent for pluripotent stem cells into neural progenitor cells of the mesencephalic floor plate region, comprising incorporating an agonist for the LPA receptor into the serum-free medium.

[0011] The present invention provides a method for efficiently obtaining nerve cells such as neural progenitor cells or dopamine neural progenitor cells from the midbrain floor plate region, a culture medium suitably used in the said method, a differentiation induction stabilizing agent for a culture medium for inducing differentiation from pluripotent stem cells to neural progenitor cells from the midbrain floor plate region, and a method for stabilizing the differentiation induction ability of a serum-free culture medium comprising chemically defined components including the differentiation induction agent from pluripotent stem cells to neural progenitor cells from the midbrain floor plate region.

[0012] Figure 1 shows a protocol (Protocol A) that is similar to the differentiation induction protocol described in Non-Patent Literature 5. AA represents ascorbic acid. Y represents Y-27632. The formulation of Protocol A without palmorfamine is called Protocol A0. The formulation of Protocol A with a palmorfamine concentration of 0.25 μM is called Protocol A1. The formulation of Protocol A with a palmorfamine concentration of 2 μM is called Protocol A2. Figure 2 shows a protocol (Protocol C) that is similar to the differentiation induction protocol (Protocol A; Figure 1) described in Non-Patent Literature 5, but with the addition of 0.1 μM of LPA from Day 0 to Day 9. The formulation of Protocol C with a palmorfamine concentration of 0.25 μM is called Protocol C1. The formulation of Protocol C with a palmorfamine concentration of 2 μM is called Protocol C2. "2xY" indicates the addition of twice the amount of Y-27632 used in Protocol A. Figure 3 shows the morphology of cells on day 2 of induction for protocols A0 (Original), A1 and A2, and protocols C1 and C2. Figure 4 shows the immunostaining images of cells on day 14 of induction for protocols A0 (Original), A1 (Pur), A2 (hPur), C1 (Pur + LPA), and C2 (hPur + LPA). Data from induction using the protocol described in Non-Patent Literature 2 (Protocol B; see Figure 8) are also presented as a control. The 1231A3 cell line was used for iPS cells. Pur: palmorfamin 0.25 μM, hPur (high-dose Pur): palmorfamin 2 μM, LPA: lysophosphatidic acid 0.1 μM. Figure 5 shows the LMX1 (LMX1A) and FOXA2 expression cell rates from the results in Figure 4. The FOXA2-expressing cell rate is the ratio of LMX1A-positive cells and FOXA-positive cells to the total number of cells (DAPI-positive cells), respectively. The same applies hereafter. Figure 6 shows the CORIN and OTX2-expressing cell rates on day 14 of induction, based on the results in Figure 4. Figure 7 shows the relative gene expression ratios of dopamine progenitor cell markers measured by qPCR in cells on day 11 of induction under protocols A0, A1, A2, C1, and C2. Data from induction using protocol B1 are also presented as a control.Figure 8 shows a differentiation induction protocol (Protocol B1) in accordance with the description in Non-Patent Document 2. Figure 9 shows a microscopic image of cells on day 7 cultured in a medium containing 0.1 μM LPA under undifferentiated iPS cell (1231A3) maintenance conditions. Y: Y-27632. Figures 10-1 and 10-2 show the relative gene expression ratios of LPA receptors measured by qPCR in cells from day 0 to 28 in Protocol A1. Figures 10-1 and 10-2 show the relative gene expression ratios of LPA receptors measured by qPCR in cells from day 0 to 28 in Protocol A1. Figure 11 shows the relative gene expression ratios of dopamine neural progenitor cell markers measured by qPCR in cells on day 11 in Protocol A1 to which LPA, LPA and VPC32183 (indicated as VPC), or NAEPA were further added from day 2 to 9. Note that differentiation induction using medium supplemented with LPA in Protocol A1 corresponds to Protocol C1. Figure 12 shows the immunohistochemical staining of cells on day 14 in Protocol A1, where LPA, LPA and VPC32183 (referred to as VPC), or NAEPA were further added on days 2 to 9. Note that differentiation induction using medium supplemented with LPA in Protocol A1 corresponds to Protocol C1. Figures 13-1 and 13-2 show the percentage of cells expressing dopamine progenitor cell markers on day 14 in Protocol A1, where LPA, LPA and VPC32183 (referred to as VPC), or NAEPA were further added on days 2 to 9. Note that differentiation induction using medium supplemented with LPA in Protocol A1 corresponds to Protocol C1. Figures 13-1 and 13-2 show the percentage of cells expressing dopamine progenitor cell markers on day 14 in Protocol A1, where LPA, LPA and VPC32183 (referred to as VPC), or NAEPA were further added on days 2 to 9. Note that differentiation induction using LPA-supplemented culture medium in Protocol A1 corresponds to Protocol C1. Figure 14 shows the relative gene expression ratios of dopamine progenitor cell markers measured by qPCR in cells on day 14 in Protocol A1 with various concentrations of LPA. The 1231A3 cell line was used (n=8). One-way ANOVA with Dunnett's multiple comparison test was performed.Figure 15 shows Protocol B1 (protocol from Figure 8, w / o LPA), Protocol D1 (w / LPA) which is Protocol B1 with added LPA, and Protocol B2 (KSR) which uses a medium without KSR. －Figure 16 shows the morphology of cells on day 2 of differentiation induction in Protocol B2 (LowPur / LowCHIR, w / o LPA) and Protocol D2 (w / LPA), in which LPA was added in Protocol B2. Protocol B1 is a protocol that conforms to the differentiation induction method described in Non-Patent Literature 2. Protocol B2 is a protocol that modifies Protocol B1 by changing the medium to one that does not contain KSR and contains B27, and further changing the concentrations of palmorfamine (Pur) and CHIR99021 (CHIR) to 0.25 μM and 0.75 μM, respectively. Figure 16 shows Protocol E, which was improved by the inventors based on Protocols A and B. Figure 17 shows the immunostaining image of cells on day 14 of differentiation induction in Protocol E. The staining results are shown in the top row from left to right for LMX1 (LMX1A), FOXA2, CORIN, and OTX2, and in the bottom row from left to right for DAPI, LMX1 (LMX1A) / FOXA2, DAPI, and CORIN / OTX2. Figure 18 shows the percentage of cells expressing dopamine progenitor cell markers on day 14 of differentiation induction in Protocol E. UK: Protocol A1 (palmorphamine 0.25 μM), JP: Protocol B1, New: Protocol E. Figure 19 shows the immunostaining images of cells on day 14 of differentiation induction in the CHIR-boost protocol in Example 8. Figure 20 shows the percentage of cells expressing each marker (total number of DAPI-positive cells) based on the immunostaining images in Figure 19 in the CHIR-boost protocol in Example 8. Figure 21 shows the culture protocol (Protocol C3) used in Example 9. Figure 22 shows the results of microarray analysis of cells on day 11 using the protocol described in Figure 21. 14,863 probe sets were analyzed, excluding low-expression genes. The results of differentiation induction in the presence and absence of LPA are compared. Circles outside the dotted line indicate genes whose expression level more than doubled with the addition of LPA. Figure 23 shows the culture protocol used in Example 10. Figure 24 shows the immunostaining images with FOXA2 and LMX1A on day 14 of cell induction of cells seeded at various seeding densities using the protocol described in Figure 23 (w / LPA or w / o LPA).Figure 25 shows the immunohistochemical staining images with CORIN and OTX2 on day 14 of induction of cells seeded at each seeding density according to the protocol (w / LPA or w / o LPA) described in Figure 23. Figures 26-1, 26-2, and 26-3 show the relative gene expression ratios of dopamine progenitor cell markers measured by qPCR on day 11 of induction of cells seeded at each seeding density according to the protocol described in Figure 23. The vertical axis represents the relative gene expression level with the expression level on Day 0 set to 1. Figures 26-1, 26-2, and 26-3 show the relative gene expression ratios of dopamine progenitor cell markers measured by qPCR on day 11 of induction of cells seeded at each seeding density according to the protocol described in Figure 23. The vertical axis represents the relative gene expression level with the expression level on Day 0 set to 1. Figures 26-1, 26-2, and 26-3 show the relative gene expression ratios of dopamine progenitor cell markers measured by qPCR on day 11 of induction of cells seeded at each seeding density in the protocol described in Figure 23. The vertical axis represents the relative gene expression level with the expression level on Day 0 set to 1. Figure 27 shows immunohistochemical staining images of brain sections in Example 11. The upper panel shows TH and HuN staining images of brain sections. The lower panel shows a magnified view of the same brain section. Upper left: HuN staining result, upper right: TH staining result, lower left: DAPI staining result, lower right: merged HuN, TH, and DAPI staining results.

[0013] I. Definitions In this specification and the claims, when a number is accompanied by the term “approximately”, it is intended to include a range of ±10% of that value. For example, “approximately 20” shall include “18 to 22”. A range of numbers includes all numbers between the two endpoints and the numbers at both endpoints. The “approximately” in relation to a range applies to both endpoints of that range. Thus, for example, “approximately 20 to 30” shall include “18 to 33”.

[0014] <Pluripotent Stem Cells> Pluripotent stem cells can be cultured in vitro. Pluripotent stem cells are stem cells that possess both the ability to differentiate into cell lineages belonging to the three germ layers (ectoderm, mesoderm, and endoderm) and / or extraembryonic tissues (pluripotency) and proliferative capacity. Pluripotent stem cells are not particularly limited. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), cultured fibroblasts, bone marrow stem cells, and pluripotent cells derived from mesenchymal stem cells (MSCs) (Muse cells). Preferred pluripotent stem cells are ES cells, ntES cells, and iPS cells.

[0015] (A) Embryonic stem cells (ES cells) ES cells are stem cells that are established from the inner cell mass of early embryos (e.g., blastocysts) of mammals such as humans and mice, and possess pluripotency and the ability to proliferate through self-renewal. ES cells are embryo-derived stem cells that originate from the inner cell mass of the blastocyst, which is the embryo after the morula stage at the 8-cell stage of the fertilized egg. ES cells have the ability to differentiate into any cell that makes up the adult body, known as pluripotency, and the ability to proliferate through self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156). Subsequently, ES cell lines were 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).

[0016] ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder or in a medium containing basic fibroblast growth factor (bFGF). Furthermore, cell maintenance through subculture can be carried out using a medium supplemented with substances such as leukemia inhibitory factor (LIF) and bFGF. For methods of establishing and maintaining human and monkey ES cells, see, for example, U.S. Patent No. 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. This is described in USA, 99:1580-1585; Klimanskaya I, et al. (2006), Nature. 444:481-485, International Publication No. 96 / 22362, International Publication No. 02 / 101057, U.S. Patent No. 5,843,780, U.S. Patent No. 6,200,806, U.S. Patent No. 6,280,718, etc.

[0017] ES cells were incubated in DMEM / F-12 medium supplemented with, for example, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 20% by volume KSR, and 4 ng / ml bFGF at 37°C and 2% by volume CO2. ２They can be maintained in a humid environment with 98% volume air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26:215-224). Furthermore, ES cells need to be passaged every 3-4 days. Passaging can be done using, for example, 1 mM CaCl ２ This can also be done using PBS containing 20% ​​by volume of KSR, 0.25% trypsin, and 0.1 mg / mL of collagenase IV.

[0018] ES cell selection can generally be performed using Real-Time PCR, with the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, and Nanog as indicators. In particular, for the selection of human ES cells, the expression of gene markers such as OCT-3 / 4, NANOG, and ECAD can be used as indicators (E. Kroon et al. (2008), Nat. Biotechnol., 26:443-452). Human ES cell lines are available by known methods. For example, WA01 (H1) and WA09 (H9) are available from the WiCell Research Institute. KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan). Human ES cells are established from human embryos within 14 days of fertilization.

[0019] (B) Spermatogonial stem cells (GS cells) Spermatogonial stem cells are pluripotent stem cells derived from the testes and are the origin for spermatogenesis. Like ES cells, spermmatogonial stem cells can be differentiated into various cell lineages. Spermatogonial stem cells have properties such as being able 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). Spermatogonial stem cells can self-replicate in a culture medium containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, spermatogonial stem cells can be obtained by repeatedly subculturing them under the same culture conditions as ES cells (Masayuki Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 41-46, Yodosha (Tokyo, Japan)).

[0020] (C) Embryonic germ cells (EG cells) Embryonic germ cells are established from primordial germ cells during the embryonic stage. Embryonic germ cells can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (Y. Matsui et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551). Embryonic germ cells are pluripotent cells similar to ES cells.

[0021] (D) Induced pluripotent stem cells (iPS cells) Induced pluripotent stem cells (iPS cells) are artificial stem cells derived from somatic cells that possess characteristics nearly equivalent to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al. Nat. Biotechnol. 26:101-106 (2008); International Publication No. 2007 / 069666; Okita, K., et al. Stem Cells 31, 458-66 (2013)). Induced pluripotent stem cells (iPS cells) can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. These reprogramming factors may consist of genes specifically expressed in ES cells, their gene products or non-coding RNAs, genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products or non-coding RNAs, or small molecule compounds.Examples of genes included in the reprogramming factors 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, dominant-negative p53, p53 gene repressors such as shRNA, EBNA1, or Glis1. These reprogramming factors may be used individually or in combination. The combination of initialization factors is as follows: International Publication Nos. 2007 / 069666, 2008 / 118820, 2009 / 007852, 2009 / 032194, 2009 / 058413, 2009 / 057831, 2009 / 075119, and 2009 / 079007. No. 2009 / 091659, No. 2009 / 101084, No. 2009 / 101407, No. 2009 / 102983, No. 2009 / 114949, 2009 / 117439, 2009 / 126250, 2009 / 126251, 2009 / 126655, No. 2009 / 157593, No. 2010 / 009015, No. 2010 / 033906, No. 2010 / 033920, No. 2010 / 04 No. 2800, No. 2010 / 050626, No. 2010 / 056831, No. 2010 / 068955, No. 2010 / 098419, No. 2 010 / 102267, 2010 / 111409, 2010 / 111422, 2010 / 115050, 2010 / 12429 No. 0, No. 2010 / 147395, No. 2010 / 147612, No. 2011 / 16588, No. 2013 / 176233, 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. Examples of combinations are given in the following publications: (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, and Maekawa M, et al. (2011), Nature. 474:225-9. Specifically, this refers to cells in which pluripotency has been induced by reprogramming differentiated somatic cells, such as fibroblasts or peripheral blood mononuclear cells, by expressing one of several combinations of genes selected from a group of reprogramming genes including OCT3 / 4, SOX2, KLF4, MYC (C-MYC, N-MYC, L-MYC), GLIS1, NANOG, SALL4, LIN28, and ESRRB. Preferred combinations of initialization factors include (1) OCT3 / 4, SOX2, KLF4, and MYC (C-MYC or L-MYC), (2) OCT3 / 4, SOX2, KLF4, LIN28, and L-MYC (Stem Cells, 2013; 31: 458-466), and (3) OCT3 / 4, SOX2, NANOG, and LIN28 (Science 2007; 318: 1917-1920).

[0022] The above-mentioned reprogramming factors include factors used to improve the establishment efficiency of histone deacetylase (HDAC) inhibitors, MEK inhibitors, glycogen synthase-3 inhibitors, DNA methyltransferase inhibitors, histone methyltransferase inhibitors, L-channel calcium agonists, butyrate, TGF-β inhibitors or ALK5 inhibitors, p53 inhibitors, ARID3A inhibitors, miRNA, Wnt signaling activators, neuropeptide Y, prostaglandins, hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2, DMRTBl, and others.

[0023] In this specification, factors used to improve the establishment efficiency are not distinguished from initialization factors. Furthermore, one or more of these may be appropriately selected and used. Examples of histone deacetylase (HDAC) inhibitors include small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, and M344; and nucleoside expression inhibitors such as siRNA and shRNA for HDAC. Examples of siRNA and shRNA for HDAC include HDAC1 siRNA Smartpool (Millipore) and HuSH 29mer shRNA Constructs against HDAC1 (OriGene). Examples of MEK inhibitors include PD184352, PD98059, U0126, SL327, and PD0325901. Examples of glycogen synthase-3 inhibitors include Bio and CHIR99021. Examples of DNA methyltransferase inhibitors include 5-azacytidinine. Examples of histone methyltransferase inhibitors include small molecule inhibitors such as BIX-01294, and nucleoside expression inhibitors such as siRNA and shRNA for Suv39hl, Suv39h2, SetDBl, and G9a. Examples of L-channel calcium agonists include Bayk8644. Examples of TGF-β inhibitors or ALK5 inhibitors include LY364947, SB431542, 616453, and A83-01. Examples of p53 inhibitors include dominant-negative compounds, siRNAs, and shRNAs for p53. Examples of ARID3A inhibitors include siRNAs and shRNAs for ARID3A. Examples of miRNAs include miR-291-3p, miR-294, miR-295, and mir-302. Examples of Wnt signaling activators include soluble Wnt3a.Examples of prostaglandins include prostaglandin E2 and prostaglandin J2. In addition to the method of producing iPS cells by direct reprogramming through gene expression, iPS cells can also be produced by inducing them from somatic cells by adding compounds (Science, 2013, 341, pp. 651-654).

[0024] There are no particular limitations on the somatic cells used in the production of iPS cells. Examples of somatic cells include tissue-derived fibroblasts, hematopoietic cells, hepatocytes, pancreatic cells, intestinal epithelial cells, and smooth muscle cells. Examples of hematopoietic cells include peripheral blood mononuclear cells (PBMCs), T cells, and umbilical cord blood-derived cells.

[0025] If the reprogramming factor is in the form of a protein, it may be introduced into somatic cells by methods such as lipofection, fusion with cell membrane-permeable peptides (e.g., HIV-derived TAT and polyarginine), microinjection, or electroporation.

[0026] When iPS cells are reprogrammed by the expression of several genes, the means of expressing the genes are not particularly limited. On the other hand, in the case of DNA, it can be introduced into somatic cells by methods such as using vectors such as viruses, plasmids, and artificial chromosomes; calcium phosphate method; lipofection method; retronectin method; electroporation; liposome method; and microinjection. Examples of viral vectors include retroviral vectors, lentiviral vectors (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, and Sendai virus vectors (International Publication No. 2010 / 008054). Artificial chromosome vectors include, for example, human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). Plasmids can be, for example, plasmid vectors or mammalian cell plasmids such as episomal vectors (Science, 322:949-953, 2008). Vectors may contain regulatory sequences such as promoters, enhancers, ribosome-binding sequences, terminators, and polyadenylation sites to enable the expression of nuclear reprogramming substances. They may also contain, if necessary, select marker sequences such as drug resistance genes (e.g., kanamycin resistance genes, ampicillin resistance genes, puromycin resistance genes, etc.), thymidine kinase genes, diphtheria toxin genes, and reporter gene sequences such as green fluorescent protein (GFP), β-glucuronidase (GUS), and FLAG. Furthermore, the above vectors may have LoxP sequences before and after the gene encoding the reprogramming factor or the promoter and the gene encoding the reprogramming factor that binds to it, in order to excise them together after introduction into somatic cells.

[0027] In the case of RNA, RNA vectors may be introduced into somatic cells by methods such as calcium phosphate, lipofection, microinjection, or electroporation. To suppress degradation, RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) may be used (Warren L, (2010) Cell Stem Cell. 7:618-630).

[0028] iPS cells can be produced in the presence or absence of feeder cells (feeder-free). When producing iPS cells in the presence of feeder cells, known methods can be used to produce iPS cells in the presence of undifferentiated maintenance factors. When producing iPS cells in the absence of feeder cells, there are no particular limitations on the culture medium used, but known maintenance media for ES cells and / or iPS cells, or media for establishing iPS cells in a feeder-free environment can be used.

[0029] Examples of culture media for iPS cell induction include DMEM containing 10-15% by volume of FBS, DMEM / F12 containing 10-15% by volume of FBS, or DMEM medium containing 10-15% by volume of FBS. These media may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, 2-mercaptoethanol, etc., as appropriate. Commercially available culture media can also be used. Commercially available culture media include feeder-required media such as mouse ES cell culture medium (TX-WES medium, ThromboX Corporation) and primate ES cell culture medium (primate ES / iPS cell medium, Reprocell Corporation), as well as feeder-free media such as serum-free media (TeSR, mTeSR, mTeSR-E8, Stemcell Technology), Essential 8 medium (E8 medium), Essential 6 medium, Stabilized Essential 8 medium, and StemFit medium (Ajinomoto Co., Inc.).

[0030] Examples of culture methods include the following: First, at 37°C, 5% CO2 by volume.２ In the presence of 10% FBS, somatic cells and reprogramming factors are brought into contact on DMEM containing 10% FBS by volume or DMEM / F12 medium containing 10% FBS by volume and cultured for approximately 4 to 7 days. After that, the cells are re-seed onto feeder cells. Examples of feeder cells include mitomycin C-treated STO cells and SNL cells. Approximately 10 days after contact between somatic cells and reprogramming factors, the cells are cultured in primate ES cell culture medium containing bFGF. iPS-like colonies can be produced approximately 30 to 45 days or more after this contact.

[0031] Alternatively, the following method can be used: First, at 37°C, 5% CO2 by volume. ２ In the presence of FBS, feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) are cultured in DMEM medium containing 10% FBS by volume. Examples of feeder cells include mitomycin C-treated STO cells and SNL cells. The DMEM medium containing 10% FBS by volume may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, 2-mercaptoethanol, etc. as appropriate. ES-like colonies can be produced after approximately 25 to 30 days or longer. Preferably, the somatic cells to be reprogrammed themselves are used instead of feeder cells (Takahashi K, et al. (2009), PLoS One. 4:e8067 or International Publication No. 2010 / 137746). Alternatively, methods using extracellular matrix (e.g., Laminin-5 (International Publication No. 2009 / 123349) and Matrigel (BD Corporation)) are exemplified.

[0032] In addition, methods using serum-free culture media are also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci US A. 106:15720-15725). Furthermore, to increase establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% to 15% by volume) (Yoshida Y, et al. (2009), Cell Stem Cell. 5:237-241 or International Publication No. 2010 / 013845).

[0033] During the above-mentioned culture, fresh medium is exchanged once a day from the second day after the start of culture. The number of somatic cells used for nuclear reprogramming is not limited, but for a 100 cm culture dish ２ it is about 5×10 ３ to about 5×10 ６ cells.

[0034] 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 genes expressed when somatic cells are reprogrammed (for example, Oct3 / 4, Nanog) is introduced as a marker gene, iPS cells established by culturing in a medium (selection medium) containing the corresponding drug can be selected. Also, when the marker gene is a fluorescent protein gene, iPS cells can be selected by observing with a fluorescence microscope, when it is a luminescent enzyme gene, by adding a luminescent substrate, and when it is a chromogenic enzyme gene, by adding a chromogenic substrate.

[0035] It is also possible to obtain established iPS cell lines. For example, human iPS cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, 1231A3 cells established at Kyoto University are available from Kyoto University or iPS Academia Japan Co., Ltd. Also, as established iPS cell lines, for example, Ff-I01 cells, Ff-I01s04 cells, QHJ-I01 and Ff-I14 cells established at Kyoto University are available from Kyoto University.

[0036] As used herein, the term "somatic cell" refers to any animal cell excluding germline cells or totipotent cells such as eggs, oocytes, and ES cells. Somatic cells are preferably mammalian cells including humans. Somatic cells include, without limitation, somatic cells of fetuses (offspring), newborns (offspring), and mature healthy or diseased somatic cells. Somatic cells also include any of primary cultured cells, subcultured cells, and established cell lines. Specifically, examples of somatic cells include (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (such as skin cells), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (such as pancreatic exocrine cells), brain cells, lung cells, kidney cells, and fat cells.

[0037] When iPS cells are used as a material for transplantation cells, from the viewpoint that rejection is less likely to occur and preferably does not occur, it is desirable to use somatic cells with the same or substantially the same HLA genotype as the recipient individual. Here, "substantially the same" means that the HLA genotypes match to such an extent that the immune response can be suppressed by an immunosuppressant against the transplanted cells. For example, somatic cells having an HLA type in which three loci of HLA-A, HLA-B, and HLA-DR or four loci including HLA-C match.

[0038] In addition, in order to suppress the immune rejection reaction when differentiated cells derived from iPS cells of other individuals are transplanted into a recipient, an iPS cell line in which a part of HLA is disrupted can also be used. For example, iPS cells in which the B2M gene or the CIITA gene is knocked out, or an iPS cell line in which the HLA-A gene, the HLA-B gene, and the CIITA gene important for the expression of HLA-Class II are knocked out can be used (see Huaigeng Xu et al, Cell Stem Cell, Vol. 24, No. 4, pp. 566-578, 2019).

[0039] (E) ES cells derived from cloned embryos obtained by nuclear transfer (ntES cells) ntES cells are ES cells derived from cloned embryos produced by nuclear transfer technology and have almost the same characteristics as ES cells derived from fertilized eggs (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). In other words, ntES (nuclear transfer ES) cells are ES cells established from the inner cell mass of a blastocyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell. To produce ntES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16:642-646) and ES cell production technology (as described above) is used (Kiyoka Wakayama et al. (2008) Experimental Medicine, Vol. 26, No. 5 (Suppl.), pp. 47-52). In nuclear transfer, an enucleated unfertilized mammalian egg can be reprogrammed by injecting the nucleus of a somatic cell into it and culturing it for several hours.

[0040] (F) Multilineage-differentiating Stress Enduring cells (Muse cells) Muse cells are pluripotent stem cells produced by the method described in International Publication No. 2011 / 007900. More specifically, Muse cells are pluripotent cells obtained by treating fibroblasts or bone marrow stromal cells with trypsin for an extended period, preferably 8 or 16 hours, followed by suspension culture, and are positive for SSEA-3 and CD105.

[0041] The pluripotent stem cells used in the methods described herein are human-derived pluripotent stem cells, preferably human iPS cells or human ES cells.

[0042] <Neural Progenitor Cells in the Midbrain Floor Plate Region> Neural progenitor cells in the midbrain floor plate region are neural progenitor cells located in the ventral part of the midbrain (floor plate), and are preferably neural progenitor cells that have a tendency to differentiate into dopamine neural progenitor cells. Examples of neural progenitor cells in the midbrain floor plate region are Foxa2, Lmx1a, Lmx1b and / or corin-positive cells. In this specification, human Foxa2 refers to polynucleotides represented by NCBI accession numbers NM_021784 or NM_153675 and the proteins encoded therein. Human Lmx1a refers to polynucleotides represented by NCBI accession numbers NM_001174069 or NM_177398 and the proteins encoded therein. Human Lmx1b refers to polynucleotides represented by NCBI accession number NM_001174147 and the proteins encoded therein. Examples of human corin include polynucleotides represented by NCBI accession number NM_006587 and the proteins encoded by them.

[0043] <Dopamine Neural Progenitor Cells> Dopamine neural progenitor cells are neural progenitor cells that have a tendency to differentiate into dopaminergic neurons. Unless otherwise specified, they may also include dopamine-producing neurons (dopaminergic neurons). In this specification, dopamine neural progenitor cells are obtained by differentiation from neural progenitor cells of the mesencephalic floor plate region. In this specification, a cell population containing dopamine neural progenitor cells may also contain other cell types, for example, a portion of which may include neural progenitor cells of the mesencephalic floor plate region.

[0044] The cell populations containing dopamine neuronal progenitor cells as used herein are preferably cell populations that do not include serotonin neurons (serotonergic neurons), GABAergic neurons (GABAergic neurons), and their progenitor cells.

[0045] The dopamine neuronal progenitor cells are preferably a cell population containing Foxa2, Nurr1, Lmx1a, Pitx3 and / or TH-positive cells. In the present invention, human Nurr1 includes the polynucleotide represented by NCBI accession number NM_006186 and the proteins encoded therein. In the present invention, human TH includes the polynucleotide represented by NCBI accession numbers NM_000360, NM_199292, or NM_199293 and the proteins encoded therein. Human Lmx1a includes the polynucleotide represented by NCBI accession number NM_001174069 or NM_177398 and the proteins encoded therein. Human Pitx3 includes the polynucleotide represented by NCBI accession number NM_005029 and the proteins encoded therein.

[0046] Methods for detecting neural progenitor cells and dopamine neural progenitor cells in the midbrain floor plate region include detecting marker genes or proteins expressed in these cells. Examples of such methods include immunohistochemistry, RT-PCR, qPCR, flow cytometry, and protein chips.

[0047] <Culture Medium> The culture medium used for culturing cells in the method described in the specification of this application may be any culture medium commonly used for culturing animal cells. Such a culture medium may be a medium prepared as a basal medium or a commercially available product. Examples of basal media include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, Neurobasal medium, or mixed media thereof, which can be used for culturing animal cells. Various culture media used in the manufacturing method described in the specification of this application can be prepared from these basal media.

[0048] <Serum-free medium> The medium used in the method described in the specification of this application is preferably a serum-free medium. In the specification of this application, "serum-free medium" means a medium that does not contain unprocessed or unpurified serum. In the specification of this application, a medium containing purified blood-derived components or animal tissue-derived components (e.g., growth factors) is also included in the definition of a serum-free medium, as long as it does not contain unprocessed or unpurified serum.

[0049] Serum-free culture media may contain serum substitutes. In this specification, serum substitutes refer to a general term for combinations of components that can obtain culture medium performance equivalent to that obtained when fetal bovine serum (FBS) or human serum is added to the basic culture medium. Examples of components used as serum substitutes include components that appropriately contain albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3'-thiolglycerol, amino acids such as glutamic acid, or equivalents thereof. Examples of the equivalents include ITS supplement (manufactured by Life Technologies) used as an FBS substitute, GlutaMax (manufactured by Life Technologies) used as a glutamic acid substitute, and Chemically-defined Lipid concentrated (manufactured by Life Technologies) used as a fatty acid. Such serum substitutes are well known to those skilled in the art and can be prepared, for example, by the method described in International Publication No. 98 / 30679. Commercially available serum substitutes may also be used. The above components can also be appropriately combined with commercially available serum substitutes. Examples of commercially available serum substitutes include Knockout Serum Replacement (manufactured by Life Technologies, now ThermoFisher Scientific; hereinafter sometimes referred to as KSR), StemSure® (manufactured by Fujifilm), Artificial Serum (KSK), B27 (manufactured by Life Technologies; https: / / www.thermofisher.com / jp / ja / home / technical-resources / media-formulation.166.html), and N2 Supplement (manufactured by Life Technologies; https: / / www.thermofisher.com / jp / ja / home / technical-resources / media-formulation.166.html).

[0050] In one embodiment, a serum-free medium is a medium consisting of chemically defined components. Here, "chemically defined medium" is not particularly limited as long as the composition is entirely chemically known, and it is not necessary for the chemical structure formula or amino acid sequence to be completely determined. A "chemically defined medium" is preferably a medium consisting only of isolated and purified components and does not contain unidentifiable or unidentified components. In one embodiment, a medium consisting of chemically defined components means a medium that does not contain components whose composition cannot be chemically determined, such as serum and conditioned medium, and is composed only of components whose composition has been chemically determined. Such a medium may be called a synthetic medium.

[0051] Culture media consisting of chemically identifiable components include media prepared by adding supplements with appropriately specified compositions to a basal medium. Examples of such supplements include B27 or N2 supplements, Chemically-defined Lipid concentrated (manufactured by Life Technologies), GlutaMax (manufactured by Life Technologies), and ITS supplements (manufactured by Life Technologies). By adding a combination of the above-mentioned supplements, it is also possible to prepare serum substitutes consisting of chemically identifiable components.

[0052] In one embodiment, the serum-free medium in this specification preferably does not contain serum substitutes such as KSR containing components whose composition is not specified or cannot be chemically specified. In one embodiment, the serum-free medium preferably does not contain biological components.

[0053] In this specification, "biological components" means components derived from living organisms that have not been isolated or purified and have not been chemically identified. In other words, even components such as proteins, sugars, and fatty acids contained in living organisms, tissues, and cells do not fall under the definition of "biological components" as used herein, such as recombinant proteins, isolated and purified compounds, or synthesized compounds. Examples of living organisms include the living organisms of mammals. However, living organisms are not limited to mammals. A culture medium containing biological substances means a culture medium containing biological substances that are unidentifiable or unidentified as substances.

[0054] In one embodiment, the serum-free medium may optionally contain one or more substances selected from fatty acids, lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvate, buffers, and inorganic salts.

[0055] In one embodiment, a serum-free medium may be used, which is obtained by adding an appropriate amount (for example, about 0.1 to 5 volume%) of commercially available B27 (manufactured by Life Technologies) to Neurobasal medium.

[0056] In one embodiment, a serum-free medium containing B27 or N2 may be used as such a serum-free medium. In one embodiment, the medium used in the method described in the specification of this application is preferably a xeno-free medium. Here, "xeno-free" means conditions in which components derived from a species different from the species of the cells to be cultured are excluded.

[0057] In the specification of the present application, feeder cells are cells other than the stem cells to be co-cultured when culturing stem cells. Examples of feeder cells include mouse fibroblasts (such as MEF), human fibroblasts, SNL cells, STO cells, etc. The feeder cells may be feeder cells that have been subjected to growth inhibition treatment. Here, examples of the growth inhibition treatment include treatment with a growth inhibitor or treatment by gamma-ray irradiation or UV irradiation. Examples of the growth inhibitor include mitomycin C. However, in the method described in the specification of the present application, it is preferable to perform the culture in the absence of feeder cells (feeder-free).

[0058] Examples of the conditions in the absence of feeder cells (feeder-free) include conditions where the above-mentioned feeder cells are not added, or conditions where they substantially do not contain feeder cells (for example, the ratio of the number of feeder cells to the total number of cells is 3% or less, preferably 0.5% or less).

[0059] Many synthetic media have been developed and commercially available for use as media for culturing pluripotent stem cells in a feeder-free manner. Examples of such synthetic media include Essential 8 (manufactured by Life Technologies), StemFit (manufactured by Ajinomoto), etc. The Essential 8 medium contains L-ascorbic acid-2-phosphate magnesium salt (64 mg / L), Selenium (14 μg / L), insulin (19.4 mg / L), NaHCO ３ (543 mg / L), transferrin (10.7 mg / L), bFGF (100 ng / mL), and a TGF-β inhibitor (TGF-β1 (2 ng / mL) or Nodal (100 ng / mL)) in DMEM / F12 medium (Nature Methods, 8, 424-429 (2011)).

[0060] <Extracellular Matrix> In the specification of this application, the extracellular matrix is ​​a supramolecular structure located outside the cell. The extracellular matrix may be of natural origin or artificial (recombinant). Examples of extracellular matrix components include collagen, proteoglycans, fibronectin, hyaluronic acid, tenascin, enterin, elastin, fibrillin, and laminin. The extracellular matrix may also be fragments of these substances. These extracellular matrix components may be a combination of two or more types, and may be cell-derived preparations such as BD Matrigel (trademark). Preferably, it is laminin or a fragment thereof. In the specification of this application, laminin is a protein having a heterotrimeric structure with one α-chain, one β-chain, and one γ-chain. Such a protein is an extracellular matrix protein in which isoforms with different subunit chain compositions exist. Laminin has about 15 isoforms, which are combinations of heterotrimers of 5 types of α-chains, 4 types of β-chains, and 3 types of γ-chains. While not particularly limited, for example, the α chain is α1, α2, α3, α4, or α5. For example, the β chain is β1, β2, β3, or β4. For example, the γ chain is γ1, γ2, or γ3. The laminin is more preferably laminin 511, consisting of α5, β1, and γ1 (Nat Biotechnol 28, 611-615 (2010)).

[0061] Laminin may be in the form of a fragment. The fragment is not particularly limited as long as it has integrin-binding activity, but for example, it may be an E8 fragment obtained by digestion with elastase (EMBO J., 3:1463-1468, 1984, J. Cell Biol., 105:589-598, 1987). Therefore, a preferred example is laminin 511E8 (preferably human laminin 511E8) described in International Publication No. 2011 / 043405, which is obtained by digesting laminin 511 with elastase. Note that laminin E8 fragments such as laminin 511E8 do not need to be elastase digestion products of laminin and may be recombinants. Laminin 511E8 is also commercially available. For example, a commercial product (iMatrix-511) can be purchased from Nippi Corporation, etc.

[0062] From the viewpoint of avoiding contamination with unidentified components, laminin or laminin fragments are preferably isolated.

[0063] <Agonists for LPA Receptors> The agonist for the LPA receptor is not particularly limited as long as it is a substance that has agonist activity against the LPA receptor, and may be a nucleic acid, a protein, or a small organic compound. Here, examples of LPA receptors include LPA1, LPA2, LPA3, LPA4, LPA5, and LPA6. In one embodiment, the LPA receptor may be any of LPA1 to LPA3 (also called EDG2, EDG4, and EDG7, respectively), and may be LPA1 or LPA2. In one embodiment, the structure of lysophosphatidic acid (LPA) is a simple structure in which the hydrogen atom at the hydroxyl group at position 1 or 2 of the glycerol skeleton is substituted with an aliphatic acyl group, an alkyl group, or an alkenyl group, and a phosphate group is bonded at position 3. LPA includes 1-acyl LPA, 1-alkyl LPA, 1-alkenyl LPA, 2-acyl LPA, etc. Among aliphatic acyl groups, alkyl groups, and alkenyl groups, aliphatic acyl groups are preferred. Examples of such aliphatic acyl groups include those having 14 to 22 carbon atoms and 1 to 4 unsaturated bonds. Examples of preferred LPAs include 18:1-LPA, 18:3-LPA, and 16:0-LPA.

[0064] As agonists for the LPA receptor, synthetic compounds well known to those skilled in the art may be used. Examples of such synthetic compounds include LPA receptor agonists described in International Publication No. 2003 / 007991 and Japanese Patent Publication No. 2008-297278. Examples of agonists for the LPA receptor include lysophosphatidic acid (LPA), specifically oleoyl-L-α-lysophosphatidic acid (1-oleoyl LPA), 20:4-lysoPA, NAEPA (CAS number: 24435-25-4), (2S)-OMPT (L-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothionate), and VPC31143. One or more of these compounds may be appropriately selected and used as agonists for the LPA receptor.

[0065] In one embodiment, the agonist for the LPA receptor may be a substance having agonist activity for at least one of LPA1 to LPA3, a substance having agonist activity for LPA1 and / or LPA2, a substance having agonist activity for LPA1, or 1-oleoil LPA.

[0066] The presence of agonist activity against the LPA receptor can be confirmed, for example, by contacting the test compound with human LPA receptor-expressing cells and then measuring the change in intracellular calcium concentration using a method such as fluorescent calcium imaging. An example of such a measurement method is the method described in Japanese Patent Application Publication No. 2008-297278.

[0067] <Substances that inhibit SMAD signaling> Substances that inhibit SMAD signaling are not particularly limited as long as they inhibit the signal transduction pathway transmitted by the Smad family. Examples of substances that inhibit SMAD signaling include substances that inhibit BMP signaling and substances that inhibit TGF-β signaling.

[0068] <BMP Inhibitors> In the specification of this application, BMP inhibitors are not particularly limited as long as they are substances that inhibit signal transduction caused by BMP, and may be nucleic acids, proteins, or small organic compounds. Here, BMPs include BMP2, BMP4, BMP7, and GDF7. Examples of BMP inhibitors include substances that act directly on BMP (e.g., antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMP (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of BMP receptors (BMPRs) to BMP, and substances that inhibit the physiological activity caused by signal transduction by BMP receptors. Examples of BMPRs include ALK2 or ALK3.As BMP signaling pathway inhibitors, compounds well known to those skilled in the art can be used, including protein inhibitors such as Chordin, Noggin, and Follistatin, and Dorsomorphin (6-[4-(2-piperidine-1-ylethoxy)phenyl]-3-pyridine-4-ylpyrazolo[1,5-a]pyrimidine) and its derivatives (PB Yu et al. (2007), Circulation, 116:II_60; PB Yu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904) and LDN193189 (4-(6-(4-(piperazine-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-yl)quinoline; 4-[6-(4-piperazine-1-ylphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinoline), LDN-214117 (1-[4-[6-methyl-5-(3,4,5-trimethyl Toxyphenyl)-3-pyridyl]phenyl]piperazine), DMH-1 (4-[6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinoline), DMH-2 (4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline), ML347 (LDN-19371 9) Examples include LDN-212854 (5-[6-[4-(piperazine-1-yl)phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline) and K02288 (3-[(6-amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol). Here, LDN193189 is well known as a BMPR (ALK2 / 3) inhibitor (hereinafter referred to as a BMPR inhibitor) and is commercially available, for example, in the form of its hydrochloride salt. Dorsomorphin and LDN193189 are available from Sigma-Aldrich and Stemgent, respectively. One or more of these may be appropriately selected and used as BMP inhibitors. The BMP inhibitor is preferably LDN193189.

[0069] <TGF-β Inhibitors> In the specification of this application, a TGF-β inhibitor is a substance that inhibits the signal transduction that leads to intracellular SMAD by binding of TGF-β to receptors on the cell membrane. TGF-β inhibitors are not particularly limited as long as they inhibit the underlying signal transduction pathway, and may be nucleic acids, proteins, or small organic compounds. Examples of TGF-β inhibitors include substances that act directly on TGF-β (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding TGF-β (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of TGF-β receptors to TGF-β, and substances that inhibit the physiological activity resulting from signal transduction by TGF-β receptors (e.g., TGF-β receptor inhibitors, Smad inhibitors, etc.). Furthermore, examples of TGF-β inhibitors include substances that inhibit binding to the ALK family receptors, or substances that inhibit the phosphorylation of SMAD by the ALK family. Examples include Lefty-1 (NCBI Accession No.: mouse: NM_010094, human: NM_020997), Lefty-2 (NCBI Accession No.: mouse: NM_177099, human: NM_003240 and NM_001172425), SB431542, SB202190 (from RKLindemann et al., Mol. Cancer, 2003). Examples include 2:20), SB505124 (GlaxoSmithKline), SB525334, GW788388, Galunicertib (LY2157299), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, RepSox (E-616452), Vactosertib (TEW-7197), LY580276 (Lilly Research Laboratories), A83-01 (International Publication No. 2009 / 146408), and their derivatives.Preferred TGF-β inhibitors include SB431542 (4-(5-benzol[1,3]dioxol-5-yl-4-pyridine-2-yl-1H-imidazole-2-yl)-benzamide) or A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), which are known inhibitors of the TGF-β receptor (ALK5) and the Activin receptor (ALK4 / 7). One or more of these may be appropriately selected and used as the TGF-β inhibitor. More preferably, the TGF-β inhibitor used in the present invention may be SB431542 or A83-01.

[0070] Furthermore, the TGF-β inhibitory activity of compounds such as SB431542 and LDN193189 can be determined by methods well known to those skilled in the art, for example, by detecting the phosphorylation of Smad using Western blotting (Mol Cancer Ther. (2004) 3, 737-45).

[0071] <Substances that activate the SHH signaling pathway> In the specification of this application, a substance that activates the SHH (Sonic hedgehog) signaling pathway is defined as a substance that causes the deinhibition of Smoothened (Smo) and subsequent activation of Gli2, which are triggered when SHH binds to its receptor, Patched (Ptch1). For example, proteins belonging to the Hedgehog family, specifically SHH or IHH (Indian Hedgehog), SHH fragments, SHH receptor agonists, Hh-Ag1.5 (Li, X., et al., Nature Biotechnology, 23, 215-221 (2005)), Smoothened Agonist, Examples include SAG (N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane; N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane), 20α-hydroxycholesterol, palmorfamine (palmorfamine, PMA; 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-nagutarenyloxy)-9H-purine-6-amine) and their derivatives (Stanton BZ, Peng LF., Mol Biosyst. 6:44-54, 2010). One or more of these may be appropriately selected and used as substances to activate the SHH signal. Examples of the SHH fragments mentioned above include recombinant Mouse Sonic Hedgehog / Shh(C25II)N-Terminus and recombinant Human Sonic Hedgehog / Shh(C24II)N-Terminus.

[0072] Preferred substances that activate the SHH signal include SHH protein (Genbank accession numbers: NM_000193, NP_000184), palmorfamine (palmorfamine), and SAG.

[0073] The activity of SHH signaling activators such as palmorfamine and SAG can be determined by methods well known to those skilled in the art, such as reporter gene assays focusing on the expression of the Gli1 gene (Oncogene (2007) 26, 5163-5168).

[0074] <Substances that activate Wnt signaling> In the specification of this application, substances that activate Wnt signaling are not particularly limited as long as they can enhance Wnt-mediated signal transduction, and may be nucleic acids, proteins, or small organic compounds. Examples of substances that activate Wnt signaling include Wnt family proteins, R-spondin 1 to 4, and GSK3β inhibitors. A GSK3β inhibitor is defined as a substance that inhibits the kinase activity of the GSK3β protein (e.g., phosphorylation ability for β-catenin). Numerous substances are already known as GSK3β inhibitors. Examples of GSK3β inhibitors include the indirubin derivative BIO (also known as GSK3β inhibitor IX; 6-bromoindirubin 3'-oxime), the maleimide derivative SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indole-3-yl)-1H-pyrrole-2,5-dione), the phenyl α-bromomethyl ketone compound GSK3β inhibitor VII (4-dibromoacetophenone), and the cell membrane-permeable phosphorylated peptide L803-mts (also known as GSK3β peptide). Inhibitors), TWS119 (3-[[6-(3-aminophenylaminophenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl]oxy]phenol), CHIR98014 (N-6-[2-[[4-(2,4-dichlorophenyl)-5-(1H-imidazole-1-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine), Tideglusib, SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1 Examples include H-pyrrole-2,5-dione), LY2090314, and the highly selective CHIR99021 (6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazole-2-yl)pyrimidine-2-ylamino]ethylamino]pyridine-3-carbonitrile). One or more of these may be appropriately selected and used as substances to activate the Wnt signal. These compounds are commercially available from companies such as Calbiochem and Biomol and are readily available.These compounds may be obtained from other sources or may be synthesized in-house. The substance that activates the Wnt signal is preferably CHIR99021.

[0075] <FGF8> In the specification of this application, FGF8 is not particularly limited. When FGF8 is human FGF8, four splicing forms are exemplified: FGF8a, FGF8b, FGF8e, or FGF8f. More preferably, FGF8b. FGF8 is commercially available from companies such as Wako and R&D Systems and is readily available. FGF8 may also be obtained by forced expression in cells by methods known to those skilled in the art.

[0076] <Neurotrophic Factors> In the specification of this application, neurotrophic factors are ligands for membrane receptors that play an important role in the survival and maintenance of function of motor neurons. For example, Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin 3 (NT-3), Neurotrophin 4 / 5 (NT-4 / 5), Neurotrophin 6 (NT-6), basic FGF (bFGF), acidic FGF, FGF-5, Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Insulin, Insulin Like Growth Factor 1 (IGF1), Insulin Like Growth Factor Examples include IGF-2 (IGF-2), Glia cell line-derived Neurotrophic Factor (GDNF), TGF-β (specifically TGF-β2, TGF-β3), Interleukin 6 (IL-6), Ciliary Neurotrophic Factor (CNTF), and LIF. Alternatively, one or more of these may be appropriately selected and used. Preferred neurotrophic factors are one or more selected from the group consisting of BDNF, GDNF, NT-3, and TGF-β3. Neurotrophic factors are readily available commercially from companies such as Wako and R&D Systems, but they may also be obtained by forced expression in cells using methods known to those skilled in the art.

[0077] <Notch Signaling Inhibitors> In the specification of this application, Notch signaling inhibitors are not particularly limited as long as they are agents capable of suppressing Notch-mediated signal transduction. Examples of Notch signaling inhibitors include γ-secretase inhibitors, anti-Notch antibodies, Notch ligand-binding site peptides (e.g., MK-0752), or substances that inhibit trimer formation by NICD, RBP-J, and WAWL in the nucleus (e.g., IWR-1, Limantrafin (CB-103), etc.). Examples of γ-secretase inhibitors include DAPT (GSI-IX), DBZ (Dibenzazepine, YO-01027), RO4929097, Semagacestat (LY450139), LY411575, FLI-06, Crenigacestat (LY3039478), and their derivatives. Among these Notch signaling inhibitors, DAPT is preferred, for example.

[0078] <ROCK Inhibitors> In the specification of this application, ROCK inhibitors are not particularly limited as long as they are agents that can suppress the function of Rho kinase (ROCK). Examples of ROCK inhibitors include Y-27632 (see, e.g., Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), Fasudil / HA1077 (see, e.g., Uenata et al., Nature 389: 990-994 (1997)), H-1152 (see, e.g., Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), and Wf-536 (see, e.g., Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324). Examples include (see 2003) and their derivatives; ripasudil; and antisense nucleic acids against ROCK, RNA interference-inducible nucleic acids (e.g., siRNA), dominant-negative variants, and their expression vectors. Furthermore, other small molecule compounds are also known as ROCK inhibitors, so such compounds or derivatives thereof can also be used in the method described in the specification of this application (see, for example, U.S. Patent Application Publications 20050209261, 20050192304, 20040014755, 20040002508, 20040002507, 20030125344, and 20030087919, as well as International Publications 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, and 2004 / 039796). One or more ROCK inhibitors may be used. The ROCK inhibitor may preferably be Y-27632.

[0079] In this specification and in the claims, "culture medium containing substance X" and "in the presence of substance X" mean a culture medium to which exogenous substance X has been added, a culture medium containing exogenous substance X, or the presence of exogenous substance X. That is, if cells or tissues present in the culture medium endogenously express, secrete, or produce substance X, endogenous substance X is distinguished from exogenous substance X, and a culture medium that does not contain exogenous substance X does not fall under the category of "culture medium containing substance X" even if it contains endogenous substance X.

[0080] II. Method for Producing Neural Progenitor Cells or Dopaminergic Neural Progenitor Cells in the Midbrain Floor Plate Region A method for producing neural progenitor cells or dopaminergic neural progenitor cells in the midbrain floor plate region includes: (1) induction of differentiation from pluripotent stem cells into neural progenitor cells in the midbrain floor plate region; (2) induction of differentiation from the neural progenitor cells in the midbrain floor plate region obtained by the differentiation induction in (1) into dopaminergic neural progenitor cells; and part or all of the differentiation induction in (1) includes culturing in a medium containing an agonist for the LPA receptor. In this specification, the start date of culture is denoted as "Day 0" or Day 0. For example, 24 hours after the start date of culture is the end of "Day 0" or "Day 0" and the start of "Day 1" or "Day 1".

[0081] <(1) Differentiation induction from pluripotent stem cells into neural progenitor cells in the mesencephalic floor plate region>

[0082] Methods for differentiating pluripotent stem cells into neural progenitor cells in the mesencephalic floor plate region are known, and for example, the methods described in Patent Documents 1-2 or Non-Patent Documents 1-4 can be used. The differentiation induction in (1) includes culturing pluripotent stem cells in a medium containing one or more substances selected from, for example, one or more substances that inhibit SMAD signaling, a substance that activates Shh signaling, and a substance that activates Wnt signaling.

[0083] Preferred pluripotent stem cells in the differentiation induction described in (1) above include iPS cells or ES cells, more preferably human iPS cells or human ES cells. There are no particular limitations on the method for producing iPS cells, and iPS cells can be produced by methods well known to those skilled in the art as described above. It is also desirable that the production of iPS cells (i.e., reprogramming somatic cells to establish pluripotent stem cells) be carried out in a feeder-free manner. There are no particular limitations on the method for obtaining ES cells, and ES cells can be produced by methods well known to those skilled in the art as described above. It is also desirable that the production of ES cells be carried out in a feeder-free manner.

[0084] The culture of pluripotent stem cells in differentiation induction described in (1) may be carried out under either suspension culture or adherent culture conditions, and is preferably carried out under adherent culture conditions. The incubator used when performing adherent culture is not particularly limited as long as it is capable of "adherent culture". Cell-adherent incubators are preferred. Examples of cell-adherent incubators include incubators whose surface has been artificially treated for the purpose of improving adhesion to cells, and specifically, incubators whose interiors are coated with a coating agent. Examples of coating agents include extracellular matrix such as laminin, enterin, collagen, gelatin, vitronectin, Synthemax (Corning), and Matrigel, as well as fragments thereof. Polymers such as polylysine and polyornithine are also suitable examples of coating agents.

[0085] Examples of laminins include laminin α5β1γ1 (hereinafter referred to as laminin 511) and laminin α1β1γ1 (hereinafter referred to as laminin 111), as well as laminin fragments (such as laminin 511E8). Furthermore, culture vessels with surface treatments such as positive charge treatment can also be used. The coating agent is preferably laminin or laminin fragment, more preferably laminin 511 or laminin 111, even more preferably laminin 511, and even more preferably laminin 511E8. Laminin 511E8 can be purchased commercially. An example of a commercially available laminin 511E8 is iMatrix-511 (Nippi Corporation). In one embodiment, the culture in step (1) may be performed on a patterning plate having multiple cell adhesion regions and non-cell adhesion regions, or it may be performed using a culture bag for suspension culture or adhesion culture.

[0086] In part or all of the differentiation induction in (1), the cells are cultured in a medium containing an agonist for the LPA receptor. Of the differentiation induction in (1), the cells are cultured in a medium containing an agonist for the LPA receptor at least until neural progenitor cells are induced from pluripotent stem cells, preferably until neural progenitor cells of the mesencephalic floor plate region can be detected from the neural progenitor cells.

[0087] In one embodiment, the "part of differentiation induction" can be described as the period from the point in time when pluripotent stem cells account for 99% or more of the total number of cells to the point in time when neural progenitor cell markers are detected. Examples of neural progenitor cell markers include NESTIN and PSA-NCAM.

[0088] In one embodiment, the "part of differentiation induction" can be described as the period from the point in time when pluripotent stem cells account for 99% or more of the total number of cells to the point in time when neural progenitor cell markers in the midbrain floor plate region are detected. Examples of neural progenitor cell markers include LMX1A, CORIN, FOXA2, and OTX2.

[0089] In one embodiment, the "part of differentiation induction" includes a period of culture in the presence of a differentiation-inducing agent necessary for differentiation induction into neurons of the midbrain floor plate region. For example, if the differentiation-inducing agent is a substance that activates the Shh signal and / or a substance that activates the Wnt signal, a culture medium containing an agonist for the LPA receptor can be used for part or all of the period of culture in the presence of the differentiation-inducing agent. In the differentiation induction in (1), for example, it is exemplified that cells are cultured in a culture medium containing an agonist for the LPA receptor on days 0 to 9, days 0 to 11, days 1 to 9, days 2 to 11, days 3 to 9, or days 3 to 11.

[0090] In differentiation induction in (1), the concentration of the agonist for the LPA receptor contained in the culture medium can be appropriately selected by those skilled in the art depending on the activity of the agonist for the LPA receptor used. For example, LPA is used at concentrations of 1 nM to 10 μM, 10 nM to 1 μM, or 80 nM to 120 nM. LPA may also be used at a concentration of approximately 100 nM. The agonist for the LPA receptor can be used in an amount that has an agonist effect on LPA receptors equivalent to that of the LPA at the above concentrations, specifically on LPA1. The agonist effect on LPA1 can be evaluated by methods such as contacting mammalian cells (e.g., CHO cells) that forcibly express the LPA receptor with the test substance and measuring the rate of increase in intracellular calcium concentration, as described above. Specifically, the agonist effect on LPA1 can be evaluated by contacting human LPA1-expressing cells with the test compound, for example, using the method described in Japanese Patent Publication No. 2008-297278 (Example 2, etc.), and then measuring the change in intracellular calcium concentration using a method such as fluorescent calcium imaging. For example, the change in intracellular calcium concentration can be measured using FLIPR (registered trademark, manufactured by Molecular Devices Inc.), and a dose-response curve can be created and calculated using the maximum fluorescence intensity over one minute of measurement.

[0091] As described above, the differentiation induction in (1) may include culturing pluripotent stem cells in a medium containing one or more substances selected from one or more substances that inhibit SMAD signaling, a substance that activates Shh signaling, and a substance that activates Wnt signaling.

[0092] (1) The concentration of the substance that inhibits the SMAD signal that may be contained in the culture medium during differentiation induction can be appropriately selected by a person skilled in the art depending on the substance that inhibits the SMAD signal used.

[0093] In the case of BMP inhibitors, for example, LDN193189 is used at concentrations of 1 nM to 10 μM, or 10 nM to 1 μM. LDN193189 may also be used at a concentration of, for example, about 100 nM. In one embodiment, the BMP inhibitor may be used in an amount that has a BMP inhibitory effect equivalent to that of LDN193189 at the aforementioned concentrations.

[0094] In the case of TGF-β inhibitors, for example, SB431542 is used at concentrations of 50 nM to 50 μM, or 500 nM to 50 μM. SB431542 may also be used at a concentration of, for example, 10 μM. A83-01 is used at concentrations of 5 nM to 50 μM, 50 nM to 5 μM, or 400 nM to 600 nM. A83-01 may also be used at a concentration of, for example, about 500 nM. In one embodiment, the TGF-β inhibitor may also be used in an amount that has the same TGF-β signaling inhibitory effect as SB431542 or A83-01 at the aforementioned concentrations.

[0095] In differentiation induction as described in (1), the concentration of the SHH signal-activating substance that may be included in the culture medium can be appropriately selected by those skilled in the art depending on the SHH signal-activating substance used. For example, Recombinant Human Sonic Hedgehog / Shh (C24II) N-Terminus (R&D, #1845-SH-100), which is the N-terminal fragment of the Shh protein, is used at concentrations of 5 ng / mL to 50 μg / mL, 50 ng / mL to 5 μg / mL, or 400 ng / mL to 800 nM. This N-terminal fragment may also be used at a concentration of, for example, about 600 ng / mL. SAG is used at concentrations of 5 nM to 50 μM, or 50 nM to 5 μM. SAG may also be used at a concentration of, for example, about 500 nM. Palmorfamine is used at concentrations of 2 nM to 200 μM, or 20 nM to 20 μM. Palmorfamine may be used at concentrations of, for example, 200 nM to 2 μM. In one embodiment, a substance that activates the SHH signal may be used in an amount that has an equivalent SHH signal activating effect to that of SAG or palmorfamine at the aforementioned concentrations.

[0096] In the differentiation induction described in (1), the concentration of the Wnt signaling activator in the culture medium can be appropriately selected by those skilled in the art depending on the type of Wnt signaling activator used. Examples of Wnt signaling activators include Wnt proteins such as Wnt3a, and GSK3β inhibitors.

[0097] For example, Wnt3a is used at concentrations of 0.1 ng / mL to 100 μg / mL, or 1 ng / mL to 50 μg / mL. Wnt3a may also be used at concentrations of, for example, 10 ng / mL to 10 μg / mL. In one embodiment, a substance that activates the Wnt signal can be used in an appropriate amount that has a Wnt signal activating effect equivalent to that of Wnt3a at the aforementioned concentrations.

[0098] In the case of GSK3β inhibitors, for example, CHIR99021 is used at concentrations of 10 nM to 100 μM, or 100 nM to 10 μM. CHIR99021 may also be used at concentrations of, for example, about 1 to 3 μM. In one embodiment, the GSK3β inhibitor can be used as appropriate in an amount that has the same GSK3β inhibitory effect as CHIR99021 at the aforementioned concentrations.

[0099] It is also preferable to increase the concentration of the Wnt signaling substance in the culture medium during cultivation. For example, the concentration of the Wnt signaling substance in the culture medium may be changed at any time during cultivation to a concentration of 1.1 to 20 times, 1.5 to 15 times, or 2 to 10 times the concentration at the start of cultivation.

[0100] If the substance that activates the Wnt signal is CHIR99021, the concentration of the Wnt signal-activating substance in the culture medium is increased to, for example, preferably 0.5 to 1.5 μM, 2 μM to 20 μM, 2.5 μM to 15 μM, or 4 μM to 10 μM.

[0101] The timing for increasing the concentration of the Wnt signaling substance in the culture medium is not particularly limited, as long as the desired effect is not impaired. For example, the timing for increasing the concentration of the Wnt signaling substance in the culture medium may be, for example, 1 to 10 days after the start of culture, 2 to 8 days after the start of culture, or 3 to 6 days after the start of culture.

[0102] In the differentiation induction described in (1), the concentration of FGF8 in the culture medium can be appropriately selected by those skilled in the art depending on the type of FGF8 used. In one embodiment, FGF8a or FGF8b can be used as FGF8. For example, FGF8b is used at concentrations of 1 ng to 1 μg / mL, 10 ng to 500 ng / mL, or 80 ng / mL to 120 ng / mL. FGF8b may also be used at a concentration of, for example, about 100 ng / mL. In another embodiment, FGF8 can be used at a concentration that has the same effect as FGF8b at the above concentrations in binding to the FGF receptor and activating its intracellular domain which has tyrosine kinase activity.

[0103] The culture medium used in differentiation induction in (1) may further contain a ROCK inhibitor at any time within 3 days, preferably within 2 days, from the start of differentiation induction in (1). The culture medium in differentiation induction in (1) may contain a ROCK inhibitor on day 1 or day 1-2 of differentiation induction in (1), for example. The concentration of the ROCK inhibitor contained in the culture medium can be appropriately selected by those skilled in the art depending on the ROCK inhibitor used. For example, Y-27632 is used at a concentration of 100 nM to 1 mM, or 1 μM to 100 μM. Y-27632 may also be used at a concentration of, for example, about 10 to 20 μM. In one embodiment, the ROCK inhibitor may be used in an amount that has a ROCK inhibitory effect equivalent to that of Y-27632 at the above concentrations.

[0104] In one embodiment, following cultivation in a medium containing the ROCK inhibitor, the cells are cultured in a medium containing an agonist for the LPA receptor. Specifically, in differentiation induction in (1), the cells are cultured in a medium containing the ROCK inhibitor for any period from the start of culture up to 48 hours, specifically 6 hours, 12 hours, 24 hours, 36 hours, 24 hours, or 48 hours, and then the medium is changed to a medium containing an agonist for the LPA receptor, and the cells are cultured in a medium containing an agonist for the LPA receptor.

[0105] The culture medium used in differentiation induction in (1) may be a serum medium or a serum-free medium, but preferably a serum-free medium that does not contain serum. The culture medium used in differentiation induction in (1) may also be a serum-free medium consisting of chemically defined components, from the viewpoint of avoiding contamination with chemically undetermined components.

[0106] In one embodiment, the differentiation induction in (1) may be carried out in the absence of KSR.

[0107] In one embodiment, the culture medium used in the differentiation induction of (1) may be a serum-free medium containing a serum substitute. The serum substitute does not contain any biological components. The differentiation induction of (1) is preferably carried out under feeder-free conditions.

[0108] When differentiation induction in (1) is performed in adherent culture, an appropriate matrix may be used as a scaffold. Cell adherent culture can be performed in a culture vessel coated with the matrix scaffold.

[0109] As a matrix that can be used as a scaffold, substances well known to those skilled in the art can be used as appropriate. Specifically, such a matrix includes the extracellular matrix described above. For example, in one embodiment, examples include laminin (Nat Biotechnol 28, 611-615 (2010)), laminin fragments (Nat Commun 3, 1236 (2012)), basement membrane preparations (Nat Biotechnol 19, 971-974 (2001)), gelatin, collagen, heparan sulfate proteoglycan, entactin, and vitronectin.

[0110] In the differentiation induction of (1), preferably, cells are cultured in a culture vessel whose surface is coated with isolated laminin 511 or E8 fragment of laminin 511, and more preferably, with E8 fragment of laminin 511.

[0111] The culture period for differentiation induction in (1) is not particularly limited, but it is preferable to obtain a cell population containing 5% or more, preferably 10% or more, of the total number of cells, of neural progenitor cells from the mesencephalic floor plate region, specifically cells positive for neural progenitor cell markers from the mesencephalic floor plate region. For example, 5 to 20 days, 5 to 15 days, 8 to 15 days, 10 to 15 days, or 11 to 14 days.

[0112] The culture conditions in differentiation induction (1), such as culture temperature and CO2 concentration, can be set as appropriate. For example, the culture temperature is preferably about 30°C to about 40°C, more preferably about 37°C. Also, for example, CO ２ The concentration is preferably about 1% by volume to about 10% by volume, more preferably about 5% by volume.

[0113] <Induction of Differentiation in (1)> There are no particular limitations on the induction of differentiation in (1), and differentiation into neural progenitor cells of the midbrain floor plate region may be induced by methods known to those skilled in the art. In one embodiment, differentiation of (1) into neurons of the midbrain floor plate region can be induced by culturing in a medium that contains one or more substances that inhibit SMAD signaling, and which may optionally contain a substance that activates Shh signaling and / or a substance that activates Wnt signaling. In this case, the medium used may contain FGF8.

[0114] In one embodiment, the differentiation induction in (1) may include culturing pluripotent stem cells in a medium containing one or more substances that inhibit SMAD signaling, a substance that activates Shh signaling, and a substance that activates Wnt signaling. In this case, the medium used in (1) may contain FGF8.

[0115] In one embodiment, the differentiation induction in (1) may satisfy three culture conditions: culturing pluripotent stem cells in a medium containing at least one SMAD signaling inhibitor, preferably a medium containing a BMP inhibitor and a TGF-β inhibitor; culturing in a medium containing a substance that activates the Shh signal within 3 days after the start of differentiation induction; and culturing in a medium containing a substance that activates the Wnt signal within 5 days after the start of differentiation induction. In this case, the medium used in (1) may contain FGF8.

[0116] Specifically, this includes the differentiation induction method described in Protocol A or Protocol B in the Examples, or a method similar thereto. In this differentiation induction method, a compound having LPA receptor agonist activity is further added to the culture medium as described above. The differentiation induction method will be described below.

[0117] <Protocol A and Similar Differentiation Induction Methods> Protocol A is based on the differentiation induction method reported in Non-Patent Document 5. The method comprises the following culture: (i) culturing pluripotent stem cells in a medium containing at least one SMAD signaling inhibitor, at least one Shh signaling activator, and at least one Wnt signaling activator.

[0118] Here, the SMAD signaling inhibitor may preferably be a combination of at least one TGF-β inhibitor and at least one BMP inhibitor.

[0119] The culture medium used in (i) is not particularly limited, but preferably may contain supplements used in the culture of nerve cells. Examples of such supplements include B27 and / or N2 supplements.

[0120] (i) is carried out for 5 to 12 days, preferably 7 to 11 days, more preferably 8 to 10 days. The culture medium used in (i) may further contain FGF8. Protocol A may further include (ii) below: (ii) After obtaining cells in the culture of (i), the cells are cultured for 1 to 8 days in a medium containing FGF8 and / or heparin. The cells obtained in the culture of (i) may be subjected to any culture operation before being used in the culture of (ii), or the cells obtained in (i) may be used directly in the culture of (ii).

[0121] <Protocol B and Similar Differentiation Induction Methods> Protocol B is based on the differentiation induction method reported in Non-Patent Literature 2. In this method, the differentiation induction in (1) includes, in this order: (i) culturing pluripotent stem cells in a medium containing one or more substances that inhibit SMAD signaling; (ii) after obtaining cells in the culture in (i), culturing the cells in a medium containing one or more substances that inhibit SMAD signaling and a substance that activates Shh signaling; and (iii) after obtaining cells in the culture in (iii), culturing the cells in a medium containing one or more substances that inhibit SMAD signaling, a substance that activates Shh signaling, and a substance that activates Wnt signaling. Protocol B may further include (iv) below: (iv) after obtaining cells in the culture in (iii), culturing the cells in a medium containing at least one SMAD signaling inhibitor and a substance that activates Wnt signaling, preferably for 1 to 7 days, more preferably for 2 to 6 days.

[0122] The cells obtained in culture (i) may be subjected to any culture operation before being used in culture (ii), or the cells obtained in (i) may be used directly in culture (ii). The cells obtained in culture (ii) may be subjected to any culture operation before being used in culture (iii), or the cells obtained in (ii) may be used directly in culture (iii). The cells obtained in culture (iii) may be subjected to any culture operation before being used in culture (iii), or the cells obtained in (iii) may be used directly in culture (iv).

[0123] Here, the SMAD signal inhibitors in (i) to (iii) may preferably be a combination of at least one TGF-β inhibitor and at least one BMP inhibitor. Alternatively, the SMAD signal inhibitor may be a BMP inhibitor.

[0124] Furthermore, the SMAD signaling inhibitor in (iv) may preferably be a BMP inhibitor.

[0125] The culture medium used in (i) to (iv) is not particularly limited, but preferably it may contain supplements used in the culture of nerve cells. Examples of such supplements include KSR, B27 and / or N2 supplements. The culture medium may also contain pyruvate, 2-mercaptoethanol, non-essential amino acids, etc.

[0126] Steps (i) to (iii) or (i) to (iv) are performed for a total of 4 to 20 days, preferably 6 to 17 days, and more preferably 8 to 15 days.

[0127] The culture medium of (ii) and / or (iii) may further contain FGF8. The concentration of FGF8 contained in the culture medium in (ii) and / or (iii) can be appropriately selected by a person skilled in the art depending on the FGF8 used, as described above.

[0128] (i) The culture period is not particularly limited, but may be, for example, 1 to 3 days, 1 to 2 days, or about 1 day.

[0129] The culture period for (ii) is not particularly limited, but may be, for example, 1 to 5 days, 1 to 4 days, 1 to 3 days, or about 1 to 2 days.

[0130] The culture period for (iii) is not particularly limited, but it is preferable to obtain a cell population containing 5% or more, preferably 10% or more, of the total number of cells, of neural progenitor cells from the mesencephalic floor plate region, specifically cells positive for neural progenitor cell markers from the mesencephalic floor plate region. For example, 1 to 10 days, 2 to 7 days, 3 to 5 days, or about 4 days.

[0131] <Improved Protocol (Protocol E and Similar Differentiation Induction Methods)> The following improved protocols are listed as methods for inducing differentiation in (1). The methods include the following (I) to (III): (I) Culturing pluripotent stem cells in a medium containing at least one SMAD signaling inhibitor, at least one Shh signaling activator, and at least one Wnt signaling activator; (II) After obtaining cells in the culture of (I), culturing the cells in a medium containing at least one SMAD signaling inhibitor and at least one Wnt signaling activator; and (III) After obtaining cells in the culture of (II), culturing the cells in a medium containing at least one SMAD signaling inhibitor. The SMAD signaling inhibitor used in the culture of (I) may preferably be a combination of at least one TGF-β inhibitor and at least one BMP inhibitor. The SMAD signaling inhibitor used in the cultures of (II) and (III) may preferably be a BMP inhibitor.

[0132] The culture medium used for differentiation induction in Protocol E is not particularly limited, but preferably may contain supplements used during the culture of nerve cells. Examples of such supplements include B27 and / or N2 supplements. For example, B27 supplement is used.

[0133] The culture of (I) is carried out for 4 to 12 days, preferably 5 to 10 days, and more preferably 6 to 8 days. The culture of (II) is carried out for 1 to 4 days, preferably 1 to 3 days. The culture of (III) is carried out for 1 to 4 days, preferably 1 to 3 days.

[0134] Any or all of the culture media used in (I), (II), and (III) may further contain FGF8.

[0135] The cultures (I) to (III) are preferably carried out for a total of 7 to 18 days, preferably 9 to 15 days or 9 to 13 days.

[0136] There are no particular limitations on the period of culture in a medium containing a substance having LPA receptor agonist activity, but the period of culture in a medium containing a substance having LPA receptor agonist activity may be the entire period of differentiation induction in (1), or it may be, for example, the culture periods of (I) and (II).

[0137] In the cultures of (I) and (II), the concentration of the Wnt signaling activator in the culture medium may be increased during the culture. For example, the concentration of the Wnt signaling activator in the culture medium may be changed to a concentration of 1.1 to 20 times the initial concentration, 1.5 to 15 times, or 2 to 10 times. The concentration may be changed in one step or gradually.

[0138] If the substance that activates the Wnt signal is CHIR99021, the concentration of the Wnt signal-activating substance in the culture medium may be, for example, 0.5 to 1.5 μM at the start of culture, and then increased to a concentration of 2 μM to 20 μM, 2.5 μM to 15 μM, or 4 μM to 10 μM.

[0139] The timing for increasing the concentration of the Wnt signaling substance in the culture medium is not particularly limited, as long as the desired effect is not impaired. For example, the timing for increasing the concentration of the Wnt signaling substance in the culture medium may be, for example, 1 to 10 days after the start of culture, 2 to 8 days after the start of culture, or 3 to 6 days after the start of culture.

[0140] In one embodiment, culture in a medium containing an agonist for the LPA receptor may be performed following culture in a medium containing the ROCK inhibitor. In differentiation induction of (1), culture can be performed in a medium containing the ROCK inhibitor for any period from the start of culture up to 48 hours, specifically, 6 hours, 12 hours, 24 hours, 36 hours, for example, 24 hours or 48 hours, and then the medium can be changed to a medium containing an agonist for the LPA receptor, and the cells can be cultured in a medium containing an agonist for the LPA receptor.

[0141] <(2) Differentiation Induction> Methods for differentiating neural progenitor cells from the midbrain floor plate region into dopaminergic neural progenitor cells are known. For example, the methods described in Non-Patent Documents 1 to 4 can be used. Differentiation induction in (2) involves culturing neural progenitor cells from the midbrain floor plate region in a medium containing, for example, neurotrophic factors and ascorbic acid or its derivatives.

[0142] (2) In differentiation induction, the culture of neural progenitor cells in the midbrain floor plate region may be carried out under either suspension culture or adherent culture conditions.

[0143] (2) The concentration of neurotrophic factors that may be included in the culture medium during differentiation induction can be appropriately selected by those skilled in the art depending on the neurotrophic factor used. BDNF can be used at concentrations of 0.2 ng / mL to 2 μg / mL, 2 ng / mL to 200 ng / mL, for example, about 20 ng / mL. GDNF can be used at concentrations of 0.1 ng / mL to 1 μg / mL, 1 ng / mL to 100 ng / mL, for example, about 10 ng / mL. TGF-β such as TGF-β3 can be used at concentrations of 0.1 ng / mL to 1 μg / mL, 0.5 ng / mL to 100 ng / mL, for example, about 1 ng / mL. In one embodiment, the neurotrophic factor can be used in an amount that has the same effect as BDNF at the above concentrations. In one embodiment, examples of combinations of neurotrophic factors include (1) BDNF and GDNF, and (2) BDNF, GDNF, and TGF-β3.

[0144] In the differentiation induction described in (2), the concentration of ascorbic acid or its derivatives that may be included in the culture medium can be appropriately selected by those skilled in the art depending on the ascorbic acid or its derivative used. For example, ascorbic acid can be used at concentrations of 2 μM to 20 mM, 20 μM to 2 mM, for example, about 200 μM. In one embodiment, derivatives of ascorbic acid can be used at concentrations equivalent to those of ascorbic acid.

[0145] The culture medium used in differentiation induction in (2) may further contain cyclo AMP (cAMP) or a derivative thereof. Examples of cAMP derivatives include dibutyryl cAMP (dbcAMP). The concentration of cAMP or its derivative contained in the culture medium can be appropriately selected by those skilled in the art depending on the cAMP or its derivative used. For example, dbcAMP is used at concentrations of 4 μM to 40 mM, 40 μM to 4 mM, for example, about 0.5 mM.

[0146] (2) The culture medium used for differentiation induction may further contain a Notch signaling inhibitor. Any substance well known to those skilled in the art can be used as the Notch signaling inhibitor, such as those listed above under <Notch signaling inhibitors>. Specifically, γ-secretase inhibitors, anti-Notch antibodies, Notch ligand-binding site peptides (e.g., MK-0752) can be used as appropriate.

[0147] In the case of γ-secretase inhibitors, for example, the concentration of the Notch signaling inhibitor contained in the culture medium can be appropriately selected by those skilled in the art depending on the γ-secretase inhibitor used. For example, DAPT is used at concentrations of 10 nM to 100 μM, 100 nM to 50 μM, or, for example, 10 μM. In one embodiment, the γ-secretase inhibitor can be used in an amount that has the same γ-secretase inhibitory effect as DAPT at the aforementioned concentrations.

[0148] (2) The culture medium used for differentiation induction may further contain FGF8. The concentration of FGF8 in the culture medium can be appropriately selected by those skilled in the art depending on the type of FGF8 used. In one embodiment, FGF8a or FGF8b can be used as FGF8. For example, FGF8b is used at concentrations of 1 ng to 1 μg / mL, 10 ng to 500 ng / mL, or 80 ng / mL to 120 ng / mL. FGF8b may also be used at a concentration of, for example, about 100 ng / mL. In another embodiment, FGF8 can be used at a concentration that has the same effect as FGF8b at the above concentrations in binding to the FGF receptor and activating its intracellular domain which has tyrosine kinase activity.

[0149] When performing differentiation induction as in (2), it is desirable to add a ROCK inhibitor to the culture medium as appropriate at the start of culture or at a time close to the start of culture. The timing of adding the ROCK inhibitor is preferably within 12 hours from the start of culture for differentiation induction as in (2), more preferably within 6 hours, even more preferably within 3 hours, even more preferably within 1 hour, and particularly preferably at the start of culture. When adding the ROCK inhibitor, it is sufficient to add it for at least 1 day, 1 to 3 days, and more preferably 1 or 2 days.

[0150] When performing differentiation induction as in (2), a culture medium containing a BMP inhibitor may be used for part or all of the culture period. In one embodiment, the cells may be cultured in a culture medium containing a BMP inhibitor for 1 to 5 days, preferably 2 to 4 days, from the start of the culture as in (2).

[0151] The culture medium used in differentiation induction in (2) may be a serum medium or a serum-free medium, but a serum-free medium is preferred. The culture medium used in differentiation induction in (2) may also be a serum-free medium consisting of chemically defined components, from the viewpoint of avoiding contamination with chemically undetermined components.

[0152] The culture vessel used when performing differentiation induction in (2) by adherent culture is not particularly limited as long as it is capable of "adherent culture," but cell-adherent culture vessels are preferred. Examples of cell-adherent culture vessels include those whose surface has been artificially treated to improve adhesion to cells, and specifically, those whose interiors are coated with a coating agent.

[0153] In the culture process for differentiation induction described in (2), an appropriate matrix may be used as a scaffold. The matrix scaffold allows for adherent culture of neural progenitor cells from the midbrain basement region in a culture vessel with a coated surface. Examples of matrices that can be used as scaffolds include laminin (Nat Biotechnol 28, 611-615 (2010)), laminin fragments (Nat Commun 3, 1236 (2012)), basement membrane preparations (Nat Biotechnol 19, 971-974 (2001)), gelatin, collagen, heparan sulfate proteoglycan, entactin, and vitronectin.

[0154] The culture vessel used when performing differentiation induction in (2) by suspension culture is not particularly limited as long as it is possible to culture the cells in a non-adherent state to the culture vessel. It can be carried out using a culture vessel that has not been artificially treated for the purpose of improving adhesion to cells, or a culture vessel that has been coated with a treatment that artificially suppresses adhesion. An example of an artificial treatment that improves adhesion to cells is coating with an extracellular matrix. Examples of materials used for treatment that suppresses adhesion include polyhydroxyethyl methacrylic acid (poly-HEMA), nonionic surfactant polyols (such as Pluronic F-127), or phospholipid-like structures. An example of a phospholipid-like structure is a water-soluble polymer (Lipidure) with 2-methacryloyloxyethyl phosphorylcholine as a constituent unit.

[0155] Following the differentiation induction in (1), the cells may be detached and dispersed as needed before the differentiation induction in (2) is performed.

[0156] In one embodiment, the cell population obtained by differentiation induction in (1) may be selected or isolated using a surface marker of the target cell as an indicator, and then differentiation induction in (2) may be performed. Known surface markers include CORIN, LRTM1, CD142, and ALCAM. Such selection or isolation can be carried out in accordance with known methods described, for example, in International Publication Nos. 2015 / 034012 and 2013 / 015457.

[0157] The culture period for differentiation induction in (2) is not particularly limited. Preferably, the culture period for differentiation induction in (2) is the period until a cell population containing 5% or more, preferably 10% or more, and more preferably 30% or more, of the total number of cells is obtained, which are dopamine neural progenitor cells, specifically, cells that are positive for dopamine neural progenitor cell markers.

[0158] The culture period for differentiation induction in (2) is not particularly limited and may be the period during which Nurr1 or Foxa2-positive cells appear. Such a culture period may be, for example, 2 to 50 days, 3 to 30 days, or 5 to 25 days.

[0159] (2) The culture for differentiation induction is preferably carried out for at least 7 days, more preferably 7 to 30 days, even more preferably 14 to 21 days, particularly preferably 14 to 16 days, and most preferably 16 days.

[0160] Culture temperature, and CO ２ The culture conditions in differentiation induction (2), such as concentration, can be set as appropriate. For example, the culture temperature is preferably about 30°C to about 40°C, more preferably about 37°C. In differentiation induction (2), CO ２ The culture is carried out in an atmosphere containing CO2. ２ CO in an atmosphere containing air ２ The concentration is preferably about 1% to about 10% by volume, more preferably about 2% to about 5% by volume.

[0161] III. Culture medium for inducing differentiation of neural progenitor cells in the mesencephalic floor plate region, differentiation induction stabilizer, and method for stabilizing differentiation induction <Culture medium> The present inventors provide a serum-free culture medium comprising chemically defined components for inducing differentiation of pluripotent stem cells into neural progenitor cells in the mesencephalic floor plate region. The culture medium contains an agonist for the LPA receptor. The culture medium may be used in the differentiation induction described in (1) above.

[0162] The above-mentioned substances can be used as appropriate agonists for the LPA receptor. Preferred agonists for the LPA receptor include lysophosphatidic acid or its salt (also known as 1-oleoyl-LPA), 20:4-lysoPA, NAEPA, (2S)-OMPT, and VPC31143.

[0163] More preferably, agonists for the LPA receptor include 1-oleoil-LPA, 20:4 lysoPA, or NAEPA.

[0164] The culture medium may further contain one or more substances selected from substances that inhibit the SMAD signal, substances that activate the Shh signal, and substances that activate the Wnt signal.

[0165] The culture medium may contain one or more substances that inhibit the SMAD signal, a substance that activates the Shh signal, and a substance that activates the Wnt signal.

[0166] <Differentiation Induction Potency Stabilizer> A differentiation induction potency stabilizer for a culture medium for inducing differentiation from pluripotent stem cells to neural progenitor cells in the mesencephalic floor plate region is provided, which includes an agonist for the LPA receptor. Here, the above-mentioned substances can be used as appropriate as the agonist for the LPA receptor. Preferred agonists for the LPA receptor include sodium lysophosphatidic acid, 1-oleoyl-LPA, 20:4-lysoPA, NAEPA, (2S)-OMPT, and VPC31143.

[0167] The form of the differentiation-inducing stabilizer is not particularly limited as long as it can be added to the culture medium. The form of the differentiation-inducing stabilizer may be liquid or solid. It is not particularly limited as a solid. The solid is typically a powder. The differentiation-inducing stabilizer may optionally contain additives to inhibit the degradation of agonists against the LPA receptor.

[0168] When the differentiation-inducing stabilizer is provided as a liquid, it is sufficient that each component contained in the differentiation-inducing stabilizer is dissolved or suspended in purified water, ethanol, dimethyl sulfoxide, or a mixture thereof. The differentiation-inducing stabilizer may be appropriately supplemented with pH adjusters, suspending agents, solubilizers, stabilizers, antioxidants, preservatives, etc., to the extent that they do not adversely affect the survival of pluripotent stem cells or nerve cells differentiated therefrom when added to the culture medium. Examples of pH adjusters include hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, monohydrogen phosphate, and sodium dihydrogen phosphate. Examples of suspending agents include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, and polyoxyethylene sorbitan monolaurate. Examples of solubilizers include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, and polyoxyethylene sorbitan monolaurate. Examples of stabilizers include sodium sulfite, sodium metasulfite, and ether. Examples of antioxidants include ascorbic acid, α-tocopherol, and glutathione. Examples of preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

[0169] <Method for stabilizing differentiation induction ability> The present inventors provide a method for stabilizing the differentiation induction ability of a serum-free medium consisting of chemically defined components, including a differentiation induction agent for pluripotent stem cells to neural progenitor cells in the mesencephalic floor plate region, which includes incorporating an agonist for the LPA receptor into the medium in part or all of the differentiation induction. The above-mentioned serum-free medium consisting of chemically defined components can be used.

[0170] The timing of adding the LPA receptor agonist to the culture medium can be adjusted as appropriate depending on the differentiation induction method, and specifically, it should follow the manufacturing method described above.

[0171] A differentiation-inducing agent that induces differentiation of pluripotent stem cells into neural progenitor cells in the mesencephalic floor plate region includes, for example, one or more substances selected from substances that inhibit one or more SMAD signals, substances that activate Shh signals, substances that activate Wnt signals, and FGF8. In one embodiment, a differentiation-inducing agent that induces differentiation of pluripotent stem cells into neural progenitor cells in the mesencephalic floor plate region includes one or more substances that inhibit SMAD signals, substances that activate Shh signals, and substances that activate Wnt signals. For further details, refer to the manufacturing method described above.

[0172] VI. Pharmaceutical Compositions and Therapeutic Methods Dopamine neural progenitor cells produced by the manufacturing method described in the specification of this application are useful as pharmaceutical compositions. The pharmaceutical composition may be a suspension in which cells are dispersed in a preservation solution or physiological saline, or a cell population containing cell aggregates containing dopamine neural progenitor cells and a preservation solution or physiological saline. The cell aggregates may be in the form of a sheet having a planar structure or in the form of spheres having a three-dimensional structure.

[0173] This pharmaceutical composition is useful as a transplantable pharmaceutical composition (graft) for patients suffering from diseases requiring the transplantation of dopamine neuron progenitor cells. The pharmaceutical composition can be used as a pharmaceutical composition for the treatment of diseases involving degeneration, damage, or dysfunction of dopamine neurons.

[0174] Examples of pharmaceutical compositions include those for the treatment of Parkinson's disease, which contain dopamine progenitor cells and / or dopamine neurons (nerve cells) obtained by the manufacturing method described in the specification of this application as an active ingredient. The number of dopamine-producing progenitor cells and / or dopamine-producing nerve cells contained in the Parkinson's disease treatment drug is not particularly limited as long as the graft can engraft after administration. For example, 1.0 × 10 per graft ４ One or more, for example, 5 x 10 ５ pieces ~ 3x10 ７ One embodiment involves transplanting individual dopamine-producing neural progenitor cells and / or dopamine-producing neurons.

[0175] In one embodiment, the pharmaceutical composition may be a pharmaceutical composition comprising dopamine neural progenitor cells produced by the manufacturing method described in the specification of this application and physiological saline.

[0176] In one embodiment, the pharmaceutical composition may be a frozen pharmaceutical composition comprising dopamine neural progenitor cells and a cryopreservation solution produced by the manufacturing method described in the specification of this application.

[0177] Furthermore, a method for treating Parkinson's disease is provided, which includes using dopamine neural progenitor cells produced by the manufacturing method described in the specification of this application, and transplanting said dopamine neural progenitor cells into the striatum of the brain of a patient requiring transplantation by stereotactic brain surgery.

[0178] The following examples illustrate the contents provided in this application in more detail, but it goes without saying that this application is not limited to these examples.

[0179] Example 1. Experiment with LPA addition 1 Differentiation induction method: [Protocol A (A0-A2) and Protocols C1, C2] In Protocol A (see Figure 1), which is based on the differentiation induction method described in Non-Patent Literature 5, differentiation induction was performed using a culture medium to which 1-oleoyl LPA (1-oleoyl-lysophosphatidic acid sodium salt / also known as 1-O-9Z-octadecanoyl-sn-glyceryl-3-phosphate, sodium salt) (CAS RN: 22556-62-3); abcam ab146430; hereinafter simply referred to as LPA in this example) was added, according to the protocol shown in Figure 2. Differentiation induction was also performed using Protocol A for comparison.

[0180] Among the human iPS cells, strain 1231A3 was received from the RIKEN Biobank as cells obtained by introducing Sox2, Oct4, Klf4, L-Myc, Lin28, Shp53, and EBNA1 into human PBMCs using an episomal vector (https: / / cellbank.brc.riken.jp / cell_bank / CellInfo / ?cellNo=HPS0381&lang=Ja). In addition, strain 404C2 was received from the Kyoto University Center for iPS Cell Research and Application as cells obtained by introducing Sox2, Oct4, Klf4, L-Myc, Lin28, and Shp53 into human fibroblasts using an episomal vector (Okita K et al. Nat Methods 8, 2011).

[0181] iPS cells were cultured according to the method described in Nakagawa M et al., Sci Rep 4, 3593, 2014. Specifically, they were maintained in undifferentiated maintenance medium (Ajinomoto; StemFiT AK02-N) containing FGF2 (bFGF) on plates coated with Laminin 511E8.

[0182] The iPS cells were dissociated using TrypLE CTS (Life Technologies). The dissociated cells were then seeded onto a plate coated with Laminin 511E8 (iMatrix-511, Nippi), which was prepared separately. Subsequently, the culture medium was replaced with differentiation medium [basic medium A containing 10 μM SB431542 (SIGMA), 0.1 μM LDN193189 (Stemgent), 1.0 μM CHIR99021 (Miltenyi Biotec), 600 ng / mL SHH (R&D), and 10 μM Y-27632 (WAKO) [DMEM / F12 (ThermoFisher) and Neurobasal Media (ThermoFisher) (mixed in a 1:1 ratio) containing 2 vol% B27 (ThermoFisher), 1 vol% N2 (ThermoFisher), and 20 μM L-Glutamine (ThermoFisher)].

[0183] Two days later (Day 2), the culture medium was changed to the same differentiation medium as on Day 0. Two days later (Day 4), the culture medium was changed to differentiation medium [basic medium B containing 10 μM SB431542, 0.1 μM LDN193189, 0.75 μM CHIR99021 and 600 ng / mL SHH (a mixture of DMEM / F12 and Neurobasal Media (1:1 ratio) containing 1 vol% B27, 0.5 vol% N2, and 20 μM L-Glutamine)]. Three days later (Day 7), the culture medium was changed to the same differentiation medium as on Day 4. Two days later (Day 9), the culture medium was replaced with differentiation medium [basic medium B containing 100 ng / mL of FGF8b (WAKO) and 1 μg / mL of heparin (Sigma)].

[0184] Two days later (Day 11), the cell population was dissociated using TripLE CTS (Life Technologies). Then, the dissociated cell population was seeded onto a plate coated with Laminin 511E8 (iMatrix-511, Nippi), which had been prepared separately. Subsequently, the culture medium was replaced with differentiation medium [Basic medium C containing 100 ng / mL FGF8b (WAKO), 1 μg / mL heparin (Sigma), 100 μg / mL BDNF (WAKO), 10 ng / mL GDNF (Peprotech), and 200 μM Ascorbic Acid (WAKO) [Neurobasal Media containing 2 vol% B27 and 20 μM L-Glutamine]]. The medium was changed with the same medium every 2-3 days during this period. The culture period varied depending on the experiment, but cells were cultured for a maximum of 35 days. Immunostaining was performed on cells on 14 days (see Figure 4). The above is referred to as Protocol A0 (Original).

[0185] Furthermore, during the period from Day 0 to Day 9 described above, groups were prepared in which either 0.25 μM palmorphamine (WAKO) or 0.1 μM LPA (abcam), or both, were added to the culture medium. Specifically, in Protocol A0, differentiation induction was carried out according to Protocol A1, in which 0.25 μM palmorphamine was added to the culture medium from Day 0 to Day 9, or Protocol A2, in which 2 μM palmorphamine was added to the culture medium. In addition, differentiation induction was carried out according to Protocol C1, in which 0.1 μM LPA was added to the culture medium from Day 0 to Day 9 in Protocol A1, and according to Protocol C2, in which 0.1 μM LPA was added to the culture medium from Day 0 to Day 9 in Protocol A2.

[0186] [Protocol B] For comparison with Protocol A (A0-A2) and Protocols C1 and C2, differentiation induction was performed using Protocol B (see Figure 8), which is based on the differentiation induction method described in Non-Patent Document 2.

[0187] Differentiation induction method: iPS cells maintained in culture using the same method as described above were dissociated using TrypLE CTS (Life Technologies). The dissociated cells were then seeded onto plates coated with Laminin 511E8 (iMatrix-511, Nippi), which were prepared separately. Subsequently, the culture medium was replaced with differentiation medium [GMEM (Gibco) containing 0.1 μM LDN193189, 0.5 μM A83-01 (WAKO), and 10 μM Y-27632, along with 8 vol% KSR (Gibco), 1 mM sodium pyruvate (Invitrogen), 0.1 mM MEM non-essential amino acid (Invitrogen), and 0.1 mM 2-mercaptoethanol (WAKO)]. On the following day (Day 1) and the day after (Day 2), the culture medium was replaced with differentiation medium [GMEM containing a basic medium containing 0.1 μM LDN193189, 0.5 μM A83-01, 2 μM palmorfamine, and 100 ng / mL FGF8b (WAKO)]. From Day 3 to Day 6, the culture medium was replaced daily with differentiation medium [GMEM containing a basic medium containing 0.1 μM LDN193189, 0.5 μM A83-01, 3 μM CHIR99021, 2 μM palmorfamine, and 100 ng / mL FGF8b (WAKO)]. From Day 7 to Day 14, the culture medium was changed daily with differentiation medium (GMEM containing a basic medium containing 0.1 μM LDN193189 and 3 μM CHIR99021).

[0188] Analysis by qPCR qPCR was performed under the following conditions. Total RNA was extracted from cells differentiated by the above differentiation induction method on day 11 using RNeasy Mini Kit or RNeasy Micro Kit (Qiagen), and cDNA was synthesized using Super Script III First-Strand Synthesis System (Invitrogen). Quantitative PCR reactions were performed in StepOne using Power SYBR Green PCR Master Mix (Applied Biosystems). Gene expression levels were evaluated using the delta-Ct method and standardized by GAPDH expression. The primer sequences used are shown in the table.

[0189]

[0190] Immunostaining Analysis Immunostaining was performed under the following conditions. Cells were fixed by treating them with 4% by volume PFA for 30 minutes. Next, the fixed cells were blocked by treating them with 2-2.5% Normal Donkey serum (NDS) / 0.3% Triton / PBS (Blocking buffer) for 60 minutes. Subsequently, the primary antibody was diluted with Blocking buffer to the specified dilution, and the resulting solution was reacted with the cells overnight at 4°C. The following day, the secondary antibody was diluted with Blocking buffer to the specified dilution, and the resulting solution was reacted with the cells at room temperature for 60 minutes. Subsequently, the cell nuclei were stained by reacting the cells with DAPI at room temperature for 10 minutes. The antibodies used in the study and their dilution concentrations are shown in the table below.

[0191]

[0192] Results: When differentiation induction was performed using Protocol A1, which involves adding 0.25 μM palmorfamine to the culture medium, in a protocol A0 that conforms to the differentiation induction method described in Non-Patent Literature 5, the induction efficiency improved (see Figure 3). Specifically, a large number of neuronal-like cells were observed in Protocol A1. This result is consistent with reports that differentiation efficiency increases with the addition of palmorfamine (see Non-Patent Literature 5). Similarly, when differentiation induction was performed using Protocol A2, which involves adding 2 μM palmorfamine, the induction efficiency also improved (Figure 3).

[0193] Next, differentiation induction was performed according to Protocol C1, which added 0.25 μM palmorfamine and 0.1 μM LPA. On the second day of induction, changes in cell morphology were observed, and compared to Protocol A0 and Protocol A1 (with palmorfamine alone), a larger number of cells with a larger adhesion area, suggesting stronger adhesion to the bottom of the dish, were observed, and the proportion of dead cells was lower (Figure 3). Similarly, when differentiation induction was performed according to Protocol C2, which added 2 μM palmorfamine and 0.1 μM LPA, a larger number of cells with a larger adhesion area were observed, and the proportion of dead cells was also low (Figure 3).

[0194] Furthermore, under the conditions of Protocol C1, which includes the addition of palmorfamine 0.25 μM and LPA 0.1 μM, the induction efficiency of midbrain dopaminergic progenitor cells was highest. Specifically, immunohistochemical analysis on day 14 of differentiation showed that LMX1A, FOXA2, and CORIN, markers of the midbrain floor plate, the origin of progenitor cells, were expressed at the highest rates under these conditions (Figures 4-6). Similar results were obtained with qPCR (Figure 7). As a control for Protocols A (A0-A2) and Protocols C1 and C2, data from samples induced using Protocol B (Figure 8) described in Non-Patent Literature 2, which uses KSR, are also presented (Figures 4-7, far right).

[0195] Discussion: It was considered that LPA contributes to improving the efficiency of differentiation induction into cells in the mesebral basement region.

[0196] Example 2. Two methods for LPA addition experiment: In Example 1, at Day 2 of the early differentiation stage, a clear increase in cell engraftment was observed under LPA addition conditions. Therefore, we evaluated whether LPA has an effect on improving the engraftment and survival of pluripotent stem cells. For the engraftment status in Example 1, please refer to the results of differentiation induction under protocols C1 and C2 in Figure 3, which add palmorfamine 0.25 μM and LPA 0.1 μM.

[0197] Specifically, iPS cells were subculturized under the conditions described in Example 1 and then cultured in AK-03N medium (AJINOMOTO) with / without Y-27632 (10 μM) and LPA 0.1 μM. Micrographs of the cells on day 7 of culture are shown in Figure 9.

[0198] Results: iPS cells could be passaged at colonial density using the ROCK inhibitor Y-27632 (Figure 9-1: Cont Y+). On the other hand, in the absence of Y-27632, apoptosis occurred and many cells died, resulting in only a very small number of colonies formed from surviving cells (Figure 9-2). In culture conditions with both Y-27632 and LPA added, LPA did not show the same iPS cell survival-promoting effect as Y-27632, and was instead confirmed to antagonize the action of Y-27632 (Figure 9-4). It was concluded that LPA does not have a survival-promoting effect on undifferentiated iPS cells (Figures 9-3 and 9-4). From the above results, no effect of improving engraftment and survival of pluripotent stem cells was observed with LPA alone. This result is not inconsistent with reports that LPA promotes Rho / ROCK signaling (Non-Patent Literature 6).

[0199] Example 3. Method for LPA1 expression: Differentiation induction was performed using protocol A1 described in Example 1, and the expression level of the LPA receptor on Day 0-28 was measured by qPCR. Two iPS cell lines, 1231A3 and 404C2, were used in this experiment.

[0200] Results: Expression of LPA1, LPA2, LPA3, LPA4, and LPA6 was observed, and when combined with the agonist and antagonist experiments described later, it was suggested that signaling mediated by at least one of LPA1, LPA2, and LPA3 is important for neural differentiation to the mesoballin region (Figure 10).

[0201] Example 4. Mechanism of LPA receptor agonist Method: The iPS cell line used was cell line 1231A3. VPC32183 (10 μM, Merck 857340P), an LPA1 / 3 antagonist, NAEPA (10 μM, (Z)-N-[2-(phosphonoxy)ethyl]-9-octadecanamide (CAS RN: 24435-25-4); Merck N0912), and LPA (100 nM, 1-oleoyl-lysophosphatidic acid sodium salt (CAS RN: 22556-62-3); abcam ab146430), an LPA1 / 2 agonist, were used.

[0202] In the protocol A1 described in Example 1, differentiation induction was performed by adding LPA, VPC32183, and / or NAEPA on Days 2-9. On Day 11, the gene expression levels of dopamine progenitor cell markers (LMX1A, FOXA2, CORIN) were evaluated by qPCR (Figure 11). In addition, the expression of dopamine progenitor cell markers (LMX1A, FOXA2, CORIN, OTX2) was evaluated by immunohistochemistry on Day 14 (Figures 12 and 13).

[0203] Results: qPCR (Figure 11) and immunohistochemical staining (Figures 12 and 13) showed that the expression levels of LMX1A, FOXA2, and CORIN increased upon addition of LPA. The increase in LMX1A and CORIN expression due to LPA addition was suppressed by co-treatment with VPC32183. Furthermore, an increase in expression was observed when NAEPA was added instead of LPA, similar to the increase with LPA. This confirms the effect of LPA receptor agonists / antagonists on the increase in LMX1A and CORIN expression.

[0204] Example 5. Method for determining the optimal concentration of LPA: The optimal concentration conditions for CHIR99021 in strain 1231A3 using protocol A1 described in Example 1 were investigated. Since it was confirmed in Example 2 that LPA antagonizes the ROCK inhibitor Y-27632, LPA was added from Day 2 instead of Day 0. LPA was investigated in the concentration range of 1 nM to 10 μM, and the expression of the dopamine neural progenitor cell marker (LMX1A) in cells on Day 14 was evaluated by immunohistochemistry. The immunohistochemistry conditions were as described in Example 1.

[0205] Results: The LMX1A expression rate was 84.7% when LPA 100 nM was added, which was significantly higher than the expression rate when LPA was not added (66.0%) (p = 0.0103). Immunocytochemistry revealed that the highest LMX1A expression rate was observed at an LPA concentration of 100 nM (Figure 14).

[0206] Example 6. Method for achieving the effect of the KSR-free protocol: In protocol B1 (Figure 8), which is based on the differentiation induction method described in Non-Patent Literature 2, differentiation induction was performed by changing from a basal medium without differentiation inducers (GMEM medium containing Pyruvate, NEAA, 2ME, and KSR; also called GMEM / KSR) to a basal medium without KSR (GMEM medium containing Pyruvate, NEAA, 2ME, and B27 and LPA; also called GMEM / B27 / LPA) (Protocol B2). Here, the concentrations of the differentiation inducers Wnt activator (CHIR99021) and Shh signaling activator (palmorfamine) were changed. Cell line 1231A3 was used as the iPS cell line.

[0207] iPS cells were cultured according to the method described in Miyazaki T et al., Nat Commun. 3:1236, 2012. Specifically, they were maintained in undifferentiated maintenance medium (Ajinomoto; StemFit AK02-N) containing FGF2 (bFGF) on Laminin 511E8 coated 6 plates.

[0208] The iPS cells were dissociated using TrypLE CTS (Life Technologies). The dissociated iPS cells were seeded onto a plate coated with Laminin 511E8 (iMatrix-511, Nippi), which was prepared separately. The culture medium was then replaced with differentiation medium [GMEM (Gibco) containing basic medium [B27, 1 mM sodium pyruvate (Invitrogen), 0.1 mM MEM non-essential amino acid (Invitrogen), and 0.1 mM 2-mercaptoethanol (WAKO)] containing LDN193189, A83-01, and Y-27632]. The following day (Day 1), the culture medium was replaced with differentiation medium (basic medium containing LDN193189, A83-01, palmorfamine, and FGF8b).

[0209] Results: In the case of Protocol D1, which added LPA to the medium of Protocol B1, no difference was observed in the degree of cell engraftment with or without LPA addition. This is thought to be because, in the protocol described in Non-Patent Literature 2, cell adhesion was good from the seeding stage due to high-density culture, and the effect of LPA was not observed (Figure 15, top).

[0210] On the other hand, in the basal medium of Protocol B1, excluding the differentiation-inducing agent, KSR was removed, CHIR99021 and palmorfamine were added at low concentrations, and cells were seeded at a low density (Protocol D2). In this case, the addition of LPA resulted in a large number of viable cells that adhered strongly to the bottom of the dish. From these results, it was found that LPA has the effect of increasing the differentiation induction efficiency (Figure 15, bottom).

[0211] Discussion: KSR exhibits variations in the composition ratio of bio-derived components from batch to batch. Therefore, batch checking is essential for KSR. De-KSR protocols, which do not use KSR, have the advantage of eliminating bio-derived components. However, in de-KSR protocols, differentiation may not occur at all depending on the differentiation induction protocol. This problem is unavoidable as long as supplements such as serum substitutes containing bio-derived components, which are difficult to rigorously chemically analyze, are used. Therefore, considering mass production requiring serum substitutes and other supplements across multiple batches, there is a concern that variations in differentiation efficiency and quality may occur when supplements containing bio-derived components are used.

[0212] The results in Figure 22 show that forebrain markers and other gene expression remained largely unchanged, but only the Floor Plate marker in the ventral midbrain showed increased gene expression upon LPA addition. Therefore, LPA does not appear to significantly alter the anterior-posterior axis site specificity or proliferative capacity in DA neuronal differentiation. It is possible that LPA alters cell adhesion, increasing viability and creating a state where various signals can easily penetrate. The use of LPA suggests the possibility of de-KSRizing the differentiation induction protocol (Protocol B) using KSR described in Non-Patent Document 2 while maintaining differentiation efficiency, and its application to industrial manufacturing methods is anticipated.

[0213] Example 7. Improved method for dopamine neural progenitor cell induction protocol: 1231A3 strains were differentiated using Protocol A (Figure 1; see Example 1, palmorfamine 0.25 μM), which is based on the differentiation induction method described in Non-Patent Literature 5; Protocol B (Figure 8), which is based on the differentiation induction method described in Non-Patent Literature 2; and the improved protocol (Protocol E; Figure 16). Evaluation was performed by immunostaining on Day 14. Additionally, 2% by volume of B27 was added to the culture medium relative to the volume of the medium. The concentrations of each factor are shown in the table below:

[0214]

[0215] The characteristics of Protocol E are: a. No KSR included, b. 500 nM of SAG used as the Shh signal activator, c. DMEM555 (manufactured by Fujifilm Wako Pure Chemical Industries, 042-30555; D-MEM / Ham's F-12 (containing L-glutamine, phenol red, HEPES, and sodium pyruvate) used as the basal medium, d. B27 added to the basal medium, e. 100 nM of LPA added on Days 2-9.

[0216] Results: Protocol E, using LPA, demonstrated effectiveness (Figures 17 and 18). Specifically, Protocol E showed the highest cell expression rates for LMX1A, FOXA2, and CORIN. Furthermore, Protocol E has the advantage of reducing protein content in the culture medium compared to Protocol B by not using FGF8b.

[0217] On the other hand, the data obtained so far suggests that LPA does not show a significant effect in differentiation induction in media containing high concentrations of differentiation-inducing factors, such as high concentrations of Shh signaling activators. Therefore, it is thought that LPA does not reduce the toxicity of the compound itself that is added as a differentiation-inducing factor. (See, for example, the addition of high concentrations of palmorfamine (hPur) in Figures 3-7).

[0218] Example 8: Method for improving the dopamine neural progenitor cell induction protocol: Using cell strain 1231A3, differentiation induction was investigated using a protocol (CHIR-boost protocol) in which the concentration of CHIR99021 added in stages was increased from Day 0 to 9, based on the improved protocol (Protocol E; Figure 16). The concentration was 1 μM from Day 0 to 3, and varied at 1 μM (control), 2.5 μM, 5.0 μM, and 7.5 μM from Day 4 to 9. In this differentiation induction, FGF8b (100 ng / mL) and heparin (1 μg / mL) were added from Day 9 to 14 in all systems. Evaluation was performed by immunostaining on Day 14. Immunostaining on Day 14 was performed in the same manner as in Example 7. The immunostained image is shown in Figure 19. Furthermore, the changes in cell percentage related to the expression of each marker, determined based on the immunostained image, are shown in Figure 20.

[0219] Results: Figures 19 and 20 show that the higher the concentration of CHIR99021 added on Days 4-9, the higher the corin positivity rate compared to control. Therefore, it was found that target cells are differentiated even in the CHIR-boost protocol using LPA.

[0220] Example 9. Method for confirming gene expression using microarrays: Differentiation induction was performed using Protocol A3 ("-LPA" in Figure 21), which is the differentiation induction method described in Non-Patent Literature 5, or in a medium to which 100 nM LPA was added in Protocol A3 (Protocol C3 ("w / LPA" in Figure 21)). Microarray analysis was performed on cells collected on Day 11. The iPS cell line used was 1231A3.

[0221] Results: Comparison of LPA+ / -: 14,863 probe sets, excluding low-expression genes, were analyzed from two lots (Figure 22). Only 21 genes showed high similarity between LPA+ and LPA-, with expression levels differing by more than twofold (Table 3). Of these 21 genes, many were found to be involved in neural differentiation (COL1A1, DDC, STMN2, STMN4). STMN2 and STMN4 are expressed in axons and growth cones and are genes involved in axon extension and neural projection. From the above, it was found that LPA addition does not affect global gene expression, but rather affects local differentiation tendencies in the midbrain and nervous system (Tables 4 and 5).

[0222] Table 4 shows the genes whose expression levels more than doubled upon addition of LPA in the microarray analysis shown in Figure 22. Note that two different values ​​are shown for NEFL and STMN2 in the table; this is the result of multiple probes being designed for the same gene.

[0223] Table 5 shows the expression levels of major gene markers related to midbrain dopamine neurons and gene markers related to the forebrain and hindbrain in the microarray analysis shown in Figure 22. Genes whose expression could not be detected are shown in gray and marked with an asterisk (*).

[0224]

[0225]

[0226] Microarray analysis confirmed that LPA addition increased the expression of ventral midbrain markers. On the other hand, no significant differences were observed in the expression levels of marker genes along the anterior-posterior axis of the brain.

[0227] COL1A1 is a marker of the vascular epithelial system and is a marker of unintended cells. A significant decrease in COL1A1 expression was observed with LPA addition, suggesting that LPA addition suppresses the formation of unintended tissues.

[0228] Example 10. Examination of culture protocol: Examination of seeded cell number and presence or absence of LPA: In a 24-well plate, 5, 10, 25, 50, 75, or 100 (x10 ４ Differentiation induction was performed using the improved protocol (Protocol E) described in Figure 23 at a seeding density of cells / well. For the study, iPS cell lines established from adult peripheral blood mononuclear cells using the Sendai virus vector (CytoTune™-2.0; ID Pharma) were used and passaged three times. Before cell detachment on Day 0 and Day 11, cells were treated with Y-27632 for 3-4 hours. The concentrations of each factor in the protocol described in Figure 23 are shown in Table 6. Cells were harvested on Day 11 and qPCR was performed. Immunofluorescence analysis (IF) was also performed on Day 14 using the method described in Example 1.

[0229]

[0230] Results: Immunofluorescence / immunocytochemistry (IF / ICC) analysis revealed that cell lines established using Sendai virus vectors tended to have higher differentiation efficiency into FOXA2, LMX1A, and CORIN-positive cells with higher seeding densities. While LPA addition increased the expression of CORIN-positive cells, no significant changes were observed in other markers (Figures 24 and 25).

[0231] At Day 11, there was no significant difference in cell viability between the groups with and without LPA addition (Table 7).

[0232] The seeding density is 25 x 10 ４In the case of cells / well, some groups could not be successfully cultured. The reason is unknown.

[0233] qPCR results showed that the expression of midbrain floorplate (FP) markers was higher with increasing seeding density (Figure 26). At all seeding densities, LPA addition increased the expression of midbrain FP markers (CORIN, LMX1A). On the other hand, no changes were observed in markers of the anterior-posterior axis of the brain (FOXA2, OTX2) with respect to seeding density or LPA addition. A tendency for the hindbrain marker (GBX2) in the untargeted region to decrease with LPA addition was observed.

[0234] The results above show a correlation between seeding density and differentiation efficiency. On the other hand, it was found that in high-density seeding, delamination may occur depending on the lot.

[0235] It was thought that the effect of LPA was to promote differentiation induction into midbrain floorplate cells, thereby maintaining differentiation efficiency regardless of culture conditions such as seeding density.

[0236] The protocol described in this specification is summarized below:

[0237] Example 11: Vivo transplantation method of dopamine neural progenitor cells in an LPA-added differentiation induction system: Cells differentiated up to Day 28 in the LPA-added protocol E (Example 7) were used. The cells were cryopreserved in a proton freezer using Bang Bang Car (trademark) hRM (GC Lymphotec Co., Ltd., Tokyo, Japan) as the cryopreservation solution. After dormancy, cell aggregates were cultured in suspension for 3 days in DMEM555 medium supplemented with CEPT cocktail, GDNF, BDNF, ascorbic acid, dbcAMP, LDN193189, FGF8, and Heparin. Approximately 200,000 cells were transplanted into each putamen of 9 12-week-old C.B-17 / Icr-scid / scidJcl mice. The CEPT cocktail is a solution prepared by mixing Chroman 50 nM, Emricasan 5 μM, and Trans ISRIB 0.7 μM in a polyethylene solution. Six months after cell transplantation, mice were perfused and fixed, and their brains were removed. Frozen sections were prepared from the obtained brain tissue. Immunostaining was performed to evaluate the expression of dopamine neuronal markers (TH) and human nuclear antigen (HuN). An example of an immunostained image is shown in Figure 27.

[0238] Results: Immunostaining confirmed cell engraftment in 8 out of 9 mice. One mouse died after 5 months (cause unknown), so brain tissue observation was not performed. As shown in Figure 27, grafts were identified from the expression of TH and HuN in brain sections (upper figure). The lower part of Figure 27 is a magnified view of the stained area of ​​the same brain section; the upper left shows the HuN staining result, the upper right shows the TH staining result, the lower left shows the DAPI staining result, and the lower right shows a merged view of the HuN and TH staining results. Multiplex staining revealed numerous cells that were co-positive for both HuN and TH. These results confirm that cells induced by this differentiation induction method engrafted in the mouse brain, matured into dopamine neurons, and remained engrafted after 6 months.

Claims

1. A method for producing neural progenitor cells or dopaminergic neural progenitor cells from pluripotent stem cells, comprising: (1) induction of differentiation from pluripotent stem cells to neural progenitor cells of the mesbrain floor plate region; (2) induction of differentiation from the neural progenitor cells of the mesbrain floor plate region obtained by the differentiation induction in (1) to dopaminergic neural progenitor cells, wherein in part or all of the differentiation induction in (1), the cells are cultured in a medium containing an agonist for LPA receptors.

2. The manufacturing method according to claim 1, wherein the culture medium used in the differentiation induction in (1) above is cultured in one or more steps in a culture medium containing one or more substances selected from one or more substances that inhibit SMAD signaling, a substance that activates Shh signaling, and a substance that activates Wnt signaling.

3. The manufacturing method according to claim 1 or 2, wherein the culture medium used in the differentiation induction in (1) above does not contain serum.

4. The manufacturing method according to claim 3, wherein the differentiation induction in (1) is carried out in the absence of knockout serum replacement (KSR).

5. The manufacturing method according to claim 3 or 4, wherein the culture medium used in the differentiation induction in (1) is a serum-free culture medium consisting of chemically defined components.

6. The manufacturing method according to any one of claims 1 to 5, wherein the agonist for the LPA receptor has agonist activity against at least one of LPA1, LPA2, and LPA3.

7. The method for producing the product according to any one of claims 1 to 6, wherein the agonist for the LPA receptor is one or more substances selected from oleoyl-L-α-lysophosphatidic acid, (1-oleoyl LPA), 20:4-lysoPA, NAEPA, (2S)-OMPT, and VPC31143.

8. The manufacturing method according to any one of claims 1 to 7, wherein the differentiation induction in (1) comprises culturing in a medium which contains one or more substances that inhibit SMAD signals and may optionally contain a substance that activates the Shh signal and / or a substance that activates the Wnt signal.

9. The manufacturing method according to claim 8, wherein the differentiation induction in (1) comprises (i) culturing in a culture medium containing one or more substances that inhibit SMAD signaling, a substance that activates Shh signaling, and a substance that activates Wnt signaling.

10. The manufacturing method according to claim 8 or 9, wherein the differentiation induction in (1) comprises culturing in a medium containing FGF8.

11. The manufacturing method according to claim 8, wherein the differentiation induction in (1) comprises: (i) culturing pluripotent stem cells in a medium comprising one or more substances that inhibit SMAD signaling and optionally a substance that activates Shh signaling and / or a substance that activates Wnt signaling; (ii) after obtaining cells in the culture in (i), culturing the cells in a medium comprising one or more substances that inhibit SMAD signaling and a substance that activates Shh signaling and may also comprise a substance that activates Wnt signaling; (iii) after obtaining cells in the culture in (ii), culturing the cells in a medium comprising one or more substances that inhibit SMAD signaling, a substance that activates Shh signaling and a substance that activates Wnt signaling, and further comprising: (iv) after obtaining cells in the culture in (iii), culturing the cells in a medium comprising one or more substances that inhibit SMAD signaling and a substance that activates Wnt signaling.

12. The manufacturing method according to claim 11, wherein the culture medium used in the cultivation of (ii) and / or (iii) is further a culture medium containing FGF8.

13. The manufacturing method according to claim 8, wherein the differentiation induction in (1) comprises: (I) culturing pluripotent stem cells in a medium containing one or more substances that inhibit SMAD signaling, a substance that activates Shh signaling, and a substance that activates Wnt signaling; (II) after obtaining cells in the culture in (I), culturing the cells in a medium containing one or more substances that inhibit SMAD signaling and a substance that activates Wnt signaling; and (III) after obtaining cells in the culture in (II), culturing the cells in a medium containing one or more substances that inhibit SMAD signaling.

14. The manufacturing method according to any one of claims 1 to 13, further comprising culturing in a medium containing a ROCK inhibitor for any period within three days from the start of differentiation induction in (1) above.

15. The method for producing the product according to claim 14, comprising culturing in a medium containing the ROCK inhibitor, followed by culturing in a medium containing an agonist for the LPA receptor.

16. The method for producing a product according to any one of claims 1 to 15, comprising the induction of differentiation from neural progenitor cells in the midbrain floor plate region of (2) to dopaminergic neural progenitor cells, wherein the culture medium used for the differentiation induction in (2) is a culture medium containing neurotrophic factors and ascorbic acid or a derivative thereof.

17. The manufacturing method according to claim 16, wherein the culture medium used for differentiation induction in (2) further comprises cAMP or a derivative thereof.

18. The method for producing neurotrophic factors according to claim 16 or 17, wherein the neurotrophic factor is one or more neurotrophic factors selected from the group consisting of BDNF, GDNF, NT-3, and TGF-β3.

19. A serum-free medium comprising chemically defined components for inducing differentiation of pluripotent stem cells into neural progenitor cells of the mesencephalic floor plate region, the medium comprising an agonist for the LPA receptor.

20. A differentiation-inducing agent for culture media containing an LPA receptor agonist, used for inducing differentiation of pluripotent stem cells into neural progenitor cells in the mesbrain floor plate region.

21. A method for stabilizing the differentiation-inducing ability of a serum-free medium comprising chemically defined components, including an agent for inducing differentiation from pluripotent stem cells to neural progenitor cells of the mesencephalic floor plate region, comprising incorporating an agonist for the LPA receptor into the serum-free medium.

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