Method for assessing differentiation potential of cells in culture broth in differentiation of pluripotent stem cells into neural cells of midbrain floor plate region

Abstract

A method for assessing the differentiation potential of cells in a culture broth in the differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region, wherein: the method for assessing the differentiation potential of cells in a culture broth in the differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region includes a step for measuring the concentration of NT-3 in culture supernatant within a culture broth obtained by culturing pluripotent stem cells in medium that contains an agent for inducing differentiation into neural cells of the midbrain floor plate region, a step for comparing the measured NT-3 concentration with a standard concentration, and a step for assessing that the cells in the culture broth can differentiate into neural cells of the midbrain floor plate region if the NT-3 concentration is equal to or greater than the standard concentration; and the culture supernatant is collected from the culture broth at any time from 48 hours to 240 hours after the start of culturing of the pluripotent stem cells.

Description

Method for determining differentiation potential of cells in culture medium in differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region

[0001] The present invention relates to a method for determining the differentiation potential of cells in a culture medium in the differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region.

[0002] Parkinson's disease is a neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Therefore, artificially producing dopaminergic neurons or dopaminergic neuronal progenitor cells by inducing differentiation from pluripotent stem cells and transplanting them into the brain of patients is expected to be an effective treatment for Parkinson's disease, and methods for producing them as cell medicines are being investigated.

[0003] On the other hand, neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family and is known to promote the survival and differentiation of neurons in the central nervous system and synapses. NT-3 is used in the process of maturing neural cells in the midbrain floor plate region, which correspond to intermediate cells, into dopaminergic neural progenitor cells when producing dopaminergic neural progenitor cells from pluripotent stem cells (Patent Document 1). Furthermore, reports have shown that the NT-3 gene is expressed in neural cells in vivo, as in Non-Patent Document 1, for example. However, until now, it has not been known whether NT-3 is involved in differentiation induction in the early stages of producing dopaminergic neural progenitor cells from pluripotent stem cells via neural progenitor cells and midbrain floor plate region neural cells. Furthermore, it has not been known whether NT-3 is secreted into the culture supernatant during the differentiation process of midbrain floor plate region neural cells.

[0004] U.S. Patent No. 7,250,294

[0005] Bernd P. , Gene Expr. 2008;14(4):241-50Frontiers in Cell and Developmental Biology, August 2020, Volume 8, Article 729

[0006] Several research groups have been studying methods for producing dopaminergic neural progenitor cells by inducing differentiation from pluripotent stem cells. However, methods for inducing differentiation of pluripotent stem cells into neural cells, such as dopaminergic neural progenitor cells, are characterized by complex procedures and the long time it takes to obtain the desired cells. Therefore, a method for producing dopaminergic neural progenitor cells is needed to predict, early in the differentiation induction process, whether the differentiation is progressing smoothly and whether a normal lot containing a certain percentage of the desired cells suitable for human transplantation will be obtained. Furthermore, a non-invasive indicator for monitoring the state of cells during the differentiation induction process is needed to confirm the normal progression of the differentiation process over time.

[0007] The present invention has been made in consideration of the above circumstances, and the problem that the present invention aims to solve is to provide a method for determining the differentiation potential into neural cells of the midbrain floor plate region, which corresponds to an intermediate state in the process of differentiation from pluripotent stem cells to dopaminergic neural progenitor cells, and which enables determination at an early stage of the differentiation induction from pluripotent stem cells to dopaminergic neural progenitor cells.

[0008] As a result of intensive research to solve the above problems, the present inventors discovered that it is possible to determine the differentiation potential of pluripotent stem cells into neural cells in the midbrain floor plate region at an early stage of differentiation induction by monitoring the concentration of neurotrophin-3 (hereinafter, sometimes referred to as "NT-3") secreted into the culture supernatant during the process of differentiation of pluripotent stem cells into dopaminergic neural progenitor cells, and thus completed the present invention.

[0009] [1] A first method of the present invention for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region is a method for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region, comprising the steps of: measuring the concentration of NT-3 in a culture supernatant of a culture medium obtained by culturing the pluripotent stem cells in a medium containing an inducer of differentiation into neural system cells of the midbrain floor plate region; comparing the measured NT-3 concentration with a reference concentration; and determining that the cells in the culture medium are capable of differentiating into neural system cells of the midbrain floor plate region when the NT-3 concentration is equal to or greater than the reference concentration, wherein the culture supernatant is collected from the culture medium any time between 48 hours and 240 hours (or 48 hours and 96 hours) after the start of culturing the pluripotent stem cells.

[0010] [2] A second method of the present invention for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region is a method for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region, comprising the steps of: measuring the concentrations of NT-3 in a first culture supernatant and a second culture supernatant in the culture medium obtained by culturing the pluripotent stem cells in a medium containing an inducer of differentiation into neural system cells of the midbrain floor plate region; determining the rate of change in NT-3 concentration from the concentrations of NT-3 in the first culture supernatant and the second culture supernatant; comparing the absolute value of the rate of change in NT-3 concentration with a standard rate of change; and determining that the cells in the culture medium are capable of differentiating into neural system cells of the midbrain floor plate region when the absolute value of the rate of change in NT-3 concentration is equal to or greater than the standard rate of change. The first culture supernatant is a culture supernatant collected from the culture medium any time between 48 hours and 192 hours (or 48 hours and 96 hours) after the start of culturing the pluripotent stem cells, and the second culture supernatant is a culture supernatant collected from the culture medium any time between 24 hours and 96 hours (or 24 hours and 72 hours) before the collection of the first culture supernatant or any time between 24 hours and 96 hours (or 24 hours and 72 hours) after the collection of the first culture supernatant.

[0011] [3] A third method of the present invention for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region, comprising the steps of: measuring the concentrations of NT-3 in a first culture supernatant, a second culture supernatant, and a third culture supernatant in the culture medium obtained by culturing the pluripotent stem cells in a medium containing an inducer of differentiation into neural system cells of the midbrain floor plate region; comparing the concentrations of NT-3 in the first culture supernatant, the second culture supernatant, and the third culture supernatant; and determining that the cells in the culture medium are capable of differentiating into neural system cells of the midbrain floor plate region when the concentration of NT-3 in the first culture supernatant is higher than the concentrations of NT-3 in the second culture supernatant and the third culture supernatant. The first culture supernatant is a culture supernatant collected from the culture medium any time between 48 hours and 192 hours (or 48 hours and 96 hours) after the start of culturing the pluripotent stem cells; the second culture supernatant is a culture supernatant collected from the culture medium any time between 24 hours and 96 hours (or 24 hours and 72 hours) before the collection of the first culture supernatant; and the third culture supernatant is a culture supernatant collected from the culture medium any time between 24 hours and 96 hours (or 24 hours and 72 hours) after the collection of the first culture supernatant.

[0012] [4] In any of the above [1] to [3], the agent for inducing differentiation into neural cells in the midbrain floor plate region preferably contains at least one SMAD signaling inhibitor.

[0013] [5] In the above [4], the SMAD signaling inhibitor preferably comprises at least one BMP signaling inhibitor and at least one TGFβ signaling inhibitor.

[0014] [6] In the above-mentioned [5], the BMP signaling inhibitor comprises at least one selected from the group consisting of LDN-193189, Noggin, DMH1, Chordin, Follistatin, K02288, LDN-214117, LDN-212854, ML347 (LDN193719) and Dorsomorphin; The TGFβ signaling inhibitors include SB-431542, A-83-01, Lefty-1, Lefty-2, Galunisertib (LY2157299), SB-202190, SB-525334, SB-505124, NPC30345, Prifenidone (S-7701), GW788388, and E-6 16452 (RepSox), SD208, TP0427736, BIBF-0775, LY3200882, Vactosertib (TEW-7197), ITD-1, SD093, SD908, LY2109761, LY364947, and LY580276. It is more preferable that the TGFβ signaling inhibitor comprises at least one selected from the group consisting of SB-431542, A-83-01, Lefty-1, Lefty-2, Galunisertib (LY2157299), SB-202190, SB-525334, SB-505124, NPC30345, Prifenidone (S-7701), GW788388, E-616452 (RepSox), SD208, ITD-1, SD093, SD908, LY2109761, LY364947, and LY580276.

[0015] [7] In any of [1] to [6] above, it is preferable that the agent for inducing differentiation into neural cells in the midbrain floor plate region further contains an SHH signaling active substance and / or a Wnt signaling active substance.

[0016] [8] In any one of the above [1] to [6], the medium preferably further contains an SHH signaling active substance and a Wnt signaling active substance.

[0017] [9] In the above-mentioned [7] or [8], the SHH signaling activator preferably comprises at least one member selected from the group consisting of SHH and fragments thereof (e.g., Shh (C24II) N-Terminus, Shh (C25II) N-Terminus) and modified forms thereof, SHH receptor, SHH receptor agonist, Hh-Ag1.5, smoothened agonist, 20a-hydroxycholesterol, purmorphamine, and SAG.

[0018]

[10] In the above [7], [8] or [9], the Wnt signaling activator preferably comprises at least one selected from the group consisting of WNT3A and a GSK-3β inhibitor.

[0019]

[11] In the above-mentioned

[10] , the GSK-3β inhibitor preferably comprises at least one selected from the group consisting of CHIR99021, BIO, CHIR98014, SKL2001, SB216763, GSK-3β inhibitor VII (4-dibromoacetophenone), and L803-mts.

[0020]

[12] In any one of [1] to

[11] above, the pluripotent stem cells are preferably iPS cells or ES cells.

[0021]

[13] The method of the present invention for producing dopaminergic neural progenitor cells or their precursor cells is a method for producing dopaminergic neural progenitor cells or their precursor cells from pluripotent stem cells, comprising: (A) starting the culture of the pluripotent stem cells under culture conditions that allow differentiation into neural cells of the midbrain floor plate region; (B) performing the method of any one of [1] to

[12] above for determining the differentiation potential of cells in the culture medium in the differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region, between 0 and 288 hours after the start of the culture of the pluripotent stem cells; and (C) deciding to continue culturing the cells in the culture medium that have been determined in step (B) to be capable of differentiating into neural cells of the midbrain floor plate region, and differentiating the cells in the culture medium into neural cells of the midbrain floor plate region. (D) culturing the neural cells in the midbrain floor plate region under culture conditions that allow differentiation into the dopaminergic neural progenitor cells, thereby differentiating the cells into the dopaminergic neural progenitor cells or their precursor cells.

[0022]

[14] In the above

[13] , the method for producing dopaminergic neural progenitor cells or their precursor cells preferably further comprises, between step (C) and step (D), a step (E) of recovering the neural cells of the midbrain floor plate region obtained in step (C).

[0023]

[15] In the method for producing dopaminergic neural progenitor cells or their precursor cells according to the above

[13] or

[14] , step (D) is preferably carried out by selecting and recovering mesencephalic floor plate cells from the neural cells of the mesencephalic floor plate region, and culturing the recovered mesencephalic floor plate cells under culture conditions that allow differentiation into the dopaminergic neural progenitor cells. That is, after appropriately differentiated mesencephalic floor plate cells, which are the target cells, are selected and recovered from a cell population of neural cells in the mesencephalic floor plate region (a cell population containing mesencephalic floor plate cells), they can be induced to differentiate into dopaminergic neural progenitor cells or their precursor cells in suspension culture or adherent culture.

[0024] The present invention provides a method for determining the differentiation potential of pluripotent stem cells into neural cells in the midbrain floor plate region, which can be determined at an early stage of differentiation induction. Furthermore, because the present invention enables non-invasive determination of differentiation potential at an early stage of differentiation induction, it also enables cost reduction by continuing differentiation culture lots with poor differentiation potential.

[0025] FIG. 1 is a graph showing the change in NT-3 concentration in the culture supernatant over time for cell groups A1 and A2 with different differentiation efficiencies as part of culture experiment A. The horizontal axis represents the number of days of culture for differentiation induction, and the vertical axis represents the NT-3 concentration in the supernatant. The data show that the NT-3 concentration increases under conditions of high differentiation induction efficiency. FIG. 2 is a graph showing the change in NT-3 concentration in the culture supernatant over time for cell groups B1 and B2 with different differentiation efficiencies as part of culture experiment B. The horizontal axis represents the number of days of culture for differentiation induction, and the vertical axis represents the NT-3 concentration in the supernatant. The data show that the NT-3 concentration increases under conditions of high differentiation induction efficiency. FIG. 3 is a graph showing the change in NT-3 concentration in the culture supernatant over time for cell groups C1 and C2 with different differentiation efficiencies as part of culture experiment C. The horizontal axis represents the number of days of culture for differentiation induction, and the vertical axis represents the NT-3 concentration in the supernatant. The data show that the NT-3 concentration increases under conditions of high differentiation induction efficiency. Figure 4 is a graph showing the results of single-cell gene expression analysis in a violin plot for cell groups D1 and D2, which differ in differentiation efficiency, as culture experiment D. The horizontal axis shows the number of days of differentiation induction culture and culture conditions, and the vertical axis shows counts per million (CPM), an index of average RNA expression level. The graph shows data indicating that many cells highly express the NT-3 gene (NTF3) under conditions of high differentiation induction efficiency. Figure 5 is a graph showing the change in NT-3 concentration in the culture supernatant over time in differentiation culture using an iPS cell line established using a Sendai virus vector as culture experiment E. The horizontal axis shows the number of days of differentiation induction culture, and the vertical axis shows the NT-3 concentration in the supernatant, indicating that the NT-3 concentration increases as differentiation progresses. Figure 6 is a graph showing the change in NT-3 concentration in the culture supernatant over time for cell groups D1 and D2, which differ in differentiation efficiency, as culture experiment D. The horizontal axis represents the number of days of culture for differentiation induction, and the vertical axis represents the NT-3 concentration in the supernatant, and the data show that the NT-3 concentration is increasing. Figure 7 is a graph showing the change in NT-3 concentration in the culture supernatant over time for cell groups F1 and F2, which were cultured under different conditions, as culture experiment F. The horizontal axis represents the number of days of culture for differentiation induction, and the vertical axis represents the NT-3 concentration in the supernatant, and the data show that the NT-3 concentration is increasing.

[0026] An embodiment of the present invention (hereinafter sometimes referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form of "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less). When no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0027] <<Method (1) for Determining the Differentiation Potential of Cells in a Culture Medium During Differentiation of Pluripotent Stem Cells into Neural System Cells of the Midbrain Floor Plate Region>> A first method for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region according to this embodiment is a method for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region, comprising the steps of: measuring the concentration of NT-3 in a culture supernatant of the culture medium obtained by culturing the pluripotent stem cells in a medium containing an inducer for differentiation into neural system cells of the midbrain floor plate region; comparing the measured NT-3 concentration with a reference concentration; and determining that the cells in the culture medium are capable of differentiating into neural system cells of the midbrain floor plate region when the NT-3 concentration is equal to or greater than the reference concentration, wherein the culture supernatant is collected from the culture medium any time between 48 and 240 hours after the initiation of culturing the pluripotent stem cells. This is described in detail below.

[0028] <Pluripotent Stem Cells> In this embodiment, "pluripotent stem cells" refer to stem cells that have pluripotency and can differentiate into all cells present in a living organism, i.e., the three germ layers (endoderm, mesoderm, and ectoderm), and also have the ability to proliferate. Examples of pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, cloned embryonic stem (ntES) cells obtained by nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, and pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells). The pluripotent stem cells are preferably at least one type selected from the group consisting of ES cells, ntES cells, and iPS cells. In one aspect of this embodiment, the pluripotent stem cells are preferably iPS cells or ES cells. The organism from which the pluripotent stem cells are derived is not particularly limited, but mammalian-derived pluripotent stem cells are preferred, and primate-derived pluripotent stem cells are more preferred. Specific examples include pluripotent stem cells derived from mice, humans, or monkeys.

[0029] (A) Embryonic Stem Cells ES cells are stem cells that have pluripotency and the ability to proliferate through self-renewal, and are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, specifically within 14 days after fertilization. ES cells are embryo-derived stem cells derived from the inner cell mass of a blastocyst, which is an embryo at the 8-cell stage of a fertilized egg, after the morula stage. ES cells have the ability to differentiate into all cells that constitute an adult, known as pluripotency, and the ability to proliferate through self-renewal. ES cells were discovered in mice in 1981 (M.J. Evans and M.H. Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in primates such as humans and monkeys (J.A. Thomson et al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; J.A. Thomson et al. (1996), Biol. Reprod., 55:254-259; J.A. Thomson and V. S. Marshall (1998), Curr. Top. Dev. Biol. , 38:133-165).

[0030] 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. Furthermore, maintenance of cells by subculture can be carried out using a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). Methods for establishing and maintaining human and monkey ES cells are described, for example, in US Pat. No. 5,843,780; Thomson JA, et al. (1995), Proc. Natl. Acad. Sci. USA. 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun. , 345:926-932;M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; H. Suemori et al. (2001), Dev. Dyn. , 222:273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I, et al. (2006), Nature. 444:481-485, etc.

[0031] As a medium for producing ES cells, for example, DMEM / F-12 medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 20% KSR (KnockOut serum replacement), and 4 ng / ml bFGF is used, and the medium is maintained at 37°C and 2% CO ２ Human ES cells can be maintained in a humid atmosphere of 98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26:215-224). ES cells must be passaged every 3 to 4 days. The passage is performed in a medium containing, for example, 1 mM CaCl ２and 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 20% ​​KSR.

[0032] ES cells can generally be selected by real-time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, and Nanog as indicators. In particular, human ES cells can be selected using the expression of gene markers such as OCT-3 / 4, NANOG, and ECAD as indicators (E. Kroon et al. (2008), Nat. Biotechnol., 26:443-452). Human ES cell lines are available from designated facilities. For example, WA01 (H1) and WA09 (H9) are available from the WiCell Research Institute. Furthermore, KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

[0033] (B) Spermatogonial stem cells Spermatogonial stem cells are pluripotent stem cells derived from the testis and are the cells that serve as the source of spermatogenesis. Like ES cells, these spermatogonial stem cells can be induced to differentiate into cells of various lineages, and have the property of being able to produce 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). These spermatogonial stem cells are capable of self-renewal in a medium containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, the above-mentioned spermatogonial stem cells can be obtained by repeated passage under the same culture conditions as ES cells (Takebayashi Masanori et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Edition), pp. 41-46, Yodosha, Tokyo, Japan).

[0034] (C) Embryonic Germ Cells Embryonic germ cells are cells established from primordial germ cells during the fetal stage and have pluripotency similar to that of ES cells. The above-mentioned 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; J.L. Resnick et al. (1992), Nature, 359:550-551).

[0035] (D) Induced pluripotent stem cells Induced pluripotent stem cells (iPS cells) can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. They are artificial stem cells derived from somatic cells that have properties similar 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); WO2007 / 069666).

[0036] The reprogramming factors may be composed of genes, their gene products, or non-coding RNAs that are specifically expressed in ES cells, or genes, their gene products, or non-coding RNAs that play an important role in maintaining the undifferentiated state of ES cells, or low-molecular-weight 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, and Glis1. These reprogramming factors may be used alone or in combination.

[0037] Combinations of reprogramming factors include those described in WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO20 09 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 03 3920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO201 0 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, HuangfuD,et al. (2008), Nat. Biotechnol. , 26:795-797, Shi Y, et al. (2008), Cell StemCell, 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, R. L. Judson et al. , (2009), Nat. Biotech. , 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci USA. 106:8912-8917, Kim JB, et al. (2009),Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9.

[0038] Specific examples of suitable combinations of reprogramming 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, 318, 1917-1920).

[0039] In addition to methods for producing induced pluripotent stem cells by direct reprogramming through gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding compounds, etc. (Science, 2013, 341, pp. 651-654).

[0040] The reprogramming factors include histone deacetylase (HDAC) inhibitors [e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC1293, and M344, and nucleic acid expression inhibitors such as siRNA and shRNA against HDAC (e.g., HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene))], MEK inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), glycogen synthase inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), and the like. kinase-3 inhibitors (e.g., Bio and CHIR99021), DNA methyltransferase inhibitors (e.g., 5-azacytidine), histone methyltransferase inhibitors (e.g., small molecule inhibitors such as BIX-01294, nucleic acid expression inhibitors such as siRNA and shRNA against Suv39hl, Suv39h2, SetDBl, and G9a), L-channel calcium Also included are factors used to enhance establishment efficiency, such as agonists (e.g., Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (e.g., LY364947, SB431542, 616453, and A-83-01), p53 inhibitors (e.g., siRNA and shRNA against p53), ARID3A inhibitors (e.g., siRNA and shRNA against ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295, and mir-302, Wnt signaling (e.g., soluble Wnt3a), neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISI, PITX2, and DMRTB1. Pluripotent stem cells as used herein also include induced pluripotent stem cells established using the above-mentioned factors used for the purpose of improving the establishment efficiency of these stem cells.

[0041] When the reprogramming factor is in the form of a protein, the reprogramming factor may be introduced into somatic cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (e.g., HIV-derived TAT and polyarginine), or microinjection.

[0042] On the other hand, when the reprogramming factor is in the form of DNA, for example, a vector such as a virus, a plasmid, or an artificial chromosome can be introduced into somatic cells by lipofection, microinjection, or the like to introduce the reprogramming factor. 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), adenoviral vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, and Sendai virus vectors (WO2010 / 008054). Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC, PAC), and the like. The plasmid may be a mammalian cell plasmid (Science, 322:949-953, 2008). The vector may contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, and polyadenylation site to enable expression of the nuclear reprogramming substance. The vector may further contain, as needed, a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a selection marker sequence such as a thymidine kinase gene or a diphtheria toxin gene, or a reporter gene sequence such as green fluorescent protein (GFP), β-glucuronidase (GUS), or FLAG. The vector may also contain 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. By doing so, after the vector is introduced into a somatic cell, the gene encoding the reprogramming factor, or the promoter and the gene encoding the reprogramming factor that binds to it, can be excised together.

[0043] Furthermore, when the reprogramming factor is in the form of RNA, the reprogramming factor may be introduced into somatic cells by techniques such as lipofection or microinjection, and RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) may be used to suppress degradation (Warren L, (2010) Cell Stem Cell. 7:618-630).

[0044] Media for establishing and maintaining iPS cells include, for example, DMEM, DMEM / F12, or DME medium containing 10 to 15% FBS, or commercially available media [e.g., a culture medium for human ES / iPS cells (AK03N, Ajinomoto Co.), a culture medium for mouse ES cells (TX-WES medium, Thrombo-X), a culture medium for primate ES cells (a culture medium for primate ES / iPS cells, ReproCell), and serum-free media (mTeSR, Stemcell Technology, Essential8, Life technologies)]. The above-mentioned DMEM, DMEM / F12, or DME medium may further contain, as appropriate, LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, 2-mercaptoethanol, etc.

[0045] Examples of the culture method include the following: First, the cells are cultured at 37°C and 5% CO ２ The somatic cells are contacted with the reprogramming factors in 10% FBS-containing DMEM or DMEM / F12 medium under an environment of 0.1% FBS and cultured for about 4 to 7 days. The somatic cells are then plated on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.), and starting about 10 days after contacting the somatic cells with the reprogramming factors, they are cultured in a bFGF-containing primate ES cell culture medium. Finally, iPS-like colonies can be generated by culturing the somatic cells for about 30 to 45 days or more after the contact.

[0046] Alternatively, 37°C, 5% CO ２In this environment, the cells are cultured on feeder cells (for example, mitomycin C-treated STO cells, SNL cells, etc.) in 10% FBS-containing DMEM medium (which may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, 2-mercaptoethanol, etc., as appropriate), and ES-like colonies can be generated after about 25 to about 30 days or more.

[0047] Desirably, iPS cells can be produced in the absence of feeder cells (feeder-free). Specific examples include methods using somatic cells to be reprogrammed instead of feeder cells (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or methods using extracellular matrices (e.g., laminin-5 (WO2009 / 123349) and Matrigel (BD)).

[0048] Other examples include a method of culturing iPS cells using a serum-free medium (Sun N, et al. (2009), Proc Natl Acad Sci USA. 106:15720-15725). Furthermore, to increase the establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5:237-241 or WO2010 / 013845).

[0049] During the culture, the medium is replaced with fresh medium once a day from the second day onward. The number of somatic cells used for reprogramming is not limited, but may be increased to 100 cells per 100 cm culture dish. ２ Approximately 5 x 10 per ３ ~Approx. 5×10 ６ Preferably in the range of cells.

[0050] iPS cells can be selected based on the shape of the colonies they form. On the other hand, if a drug resistance gene that is expressed in conjunction with a gene (e.g., Oct3 / 4, Nanog) that is expressed when somatic cells are reprogrammed is introduced as a marker gene, established iPS cells can be selected by culturing them in a medium (selective medium) containing the corresponding drug. Furthermore, if the marker gene is a fluorescent protein gene, iPS cells can be selected by observing them under a fluorescent microscope. If the marker gene is a luciferase gene, iPS cells can be selected by adding a luminescent substrate. Furthermore, if the marker gene is a chromogenic enzyme gene, iPS cells can be selected by adding a chromogenic substrate.

[0051] It is also possible to obtain established induced pluripotent stem cell lines; for example, human induced pluripotent stem cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells established at Kyoto University are available from Kyoto University and iPS Academia Japan, Inc. Examples of established induced pluripotent stem cell lines include Ff-I01 cells, Ff-I14 cells, and QHJI01s04 cells established at Kyoto University and available from Kyoto University. iPS cells can also be produced using, for example, somatic cells.

[0052] As used herein, the term "somatic cells" refers to any animal cell (preferably a mammalian cell, including a human cell) excluding germline cells such as eggs, oocytes, and ES cells, or pluripotent stem cells. Somatic cells include, but are not limited to, fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature, healthy or diseased somatic cells. Somatic cells also include primary culture cells, passaged cells, and established cell lines. Specifically, somatic cells include, for example, (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 (skin cells, etc.), hair cells, liver cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0053] Furthermore, when iPS cells are used as a source of transplantation cells, it is desirable to use somatic cells with the same or substantially the same HLA genotype as the recipient individual, from the viewpoint of suppressing rejection after transplantation. Here, "substantially the same" means that the HLA genotype matches the transplanted cells to an extent that immune responses can be suppressed with an immunosuppressant, for example, somatic cells with an HLA type that matches the three gene loci of HLA-A, HLA-B, and HLA-DR, or the four gene loci of HLA-A, HLA-B, HLA-DR, and HLA-C.

[0054] (E) ES Cells Derived from Cloned Embryos Obtained by Nuclear Transfer Somatic cell-derived ES cells (nt ES cells) are ES cells derived from cloned embryos produced by nuclear transfer technology, and have almost the same properties 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). That is, nt ES (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 nt ES cells, a combination of nuclear transfer technology (J.B. Cibelli et al. (1998), Nature Biotechnol., 16:642-646) and ES cell production technology (mentioned above) is used (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, followed by culturing for several hours to allow reprogramming.

[0055] (F) Multilineage-differentiating Stress Enduring Cells (Muse Cells) Muse cells are pluripotent stem cells produced by the method described in WO 2011 / 007900. More specifically, Muse cells are pluripotent cells obtained by trypsinizing fibroblasts or bone marrow stromal cells for a long period of time, preferably 8 or 16 hours, followed by suspension culture, and are SSEA-3 and CD105 positive cells.

[0056] <Nervous System Cells> In this specification, the term "neural cells" refers to any type of nervous system cell, such as central nervous system cells, peripheral nervous system cells, autonomic nervous system cells, motor nervous system or sensory system nervous system cells, midbrain floor plate nervous system cells, and stem or progenitor cells thereof.

[0057] <Dopamine neural progenitor cells> As used herein, "dopamine neural progenitor cells" refers to precursor cells of dopaminergic neurons that are destined to differentiate into dopaminergic neurons (also referred to as "dopamine neurons" or "dopaminergic neurons"). The dopaminergic neural progenitor cells are FOXA2-positive and βIII Tubulin (TUJ1)-positive, and preferably may be cells (also referred to as "positive cells") that further express one or more genes and proteins (differentiation markers) selected from the group consisting of OTX2, CORIN, CD142, LMX1A, LMX1B, EN1, Nurr1, PITX3, DAT, GIRK2, and TH. Cells that express one or a combination of several of these genes and that can be induced to differentiate into dopaminergic neurons can be determined to be dopaminergic neural progenitor cells. As used herein, a cell population containing dopaminergic neural progenitor cells may also contain dopaminergic neurons. The cell population containing dopaminergic neuronal progenitor cells herein is preferably a cell population that does not contain serotonergic neurons.

[0058] The cell population containing dopaminergic neural progenitor cells is preferably a cell population containing cells expressing one or more proteins selected from the group consisting of FOXA2, βIII Tubulin (TUJ1), OTX2, CORIN, CD142, LMX1A, LMX1B, EN1, Nurr1, PITX3, DAT, GIRK2, and TH. As described below, dopaminergic neural progenitor cells can be induced to differentiate from pluripotent stem cells using a mechanism common to in vivo development. First, neural progenitor cells are induced by inducing pluripotent stem cells into neural cells (neural induction). The neural progenitor cells are characterized by being, for example, SOX1-positive and / or NESTIN-positive. The neural progenitor cells are then induced to differentiate into midbrain floor plate cells, which are intermediate cells. Specifically, they are induced to differentiate into mesencephalic floor plate cells (described later) via precursor cells of mesencephalic floor plate cells. Mesencephalic floor plate cells are also called midbrain floor plate cells, and correspond to the precursor cells of dopaminergic neural progenitors that are destined to differentiate into mesencephalic dopaminergic neural progenitor cells.

[0059] <Nervous System Cells of the Midbrain Floor Plate Region> The "midbrain floor plate region" or "midbrain floor plate" is a region that appears only at a certain stage during the development of the mammalian fetal brain and spinal cord. Specifically, the fetal brain is divided into the forebrain, midbrain, and hindbrain along its anterior-posterior axis, and the floor plate is the most ventral region along its dorso-ventral axis. Dopaminergic neurons (also called dopaminergic neurons) are known to develop from the midbrain floor plate region. Specifically, even when differentiation of dopaminergic neurons or their precursor cells, dopaminergic neural progenitor cells, is induced from pluripotent stem cells in vitro, they are generated via nervous system cells in the midbrain floor plate region. In other words, in this embodiment, "neural cells of the midbrain floor plate region" refer to cells that are classified as neural cells among cells that appear in the midbrain floor plate region during development and that can differentiate into dopaminergic neural progenitor cells and further into dopaminergic neurons (dopamine-producing neurons), and include the midbrain floor plate cells described above. As used herein, the neural cells of the midbrain floor plate region are neural cells of the midbrain floor plate region that have been induced to differentiate from the pluripotent stem cells in an in vitro system. Examples of the neural cells of the midbrain floor plate region or midbrain floor plate cells include cells positive for one or more genes and proteins selected from the group consisting of CORIN, LRTM1, LMX1A, FOXA2, NGN2, DDC, OTX2, LMX1B, and CD142. Specifically, examples of such cells include CORIN-positive cells, LRTM1-positive cells, cells positive for both FOXA2 and LMX1A, and cells positive for FOXA2 and one or more selected from the group consisting of CORIN, LRTM1, NGN2, DDC, OTX2, LMX1A, LMX1B, and CD142. In this embodiment, the cell population containing neural cells of the midbrain floor plate region or midbrain floor plate cells is preferably a population containing FOXA2-positive cells, and desirably further contains cells expressing one or more genes and proteins selected from the group consisting of CORIN, LRTM1, OTX2, NGN2, DDC, LMX1A, LMX1B, and CD142. The cell population containing neural cells of the midbrain floor plate region or midbrain floor plate cells may also contain dopaminergic neural progenitor cells.The cell population containing neural cells of the midbrain floor plate region herein is preferably a cell population containing cells positive for one or more proteins selected from the group consisting of CORIN, LRTM1, FOXA2, LMX1A, LMX1B, and CD142.

[0060] <Marker-Positive Cells> In this embodiment, the term "marker-positive cells" refers to cells in which a specific marker protein is expressed on the cell surface or intracellularly in an amount that can be recognized by an antibody against the marker protein.

[0061] In this embodiment, "Corin-positive cells" refer to cells expressing Corin protein on their cell surface in an amount that can be recognized by an anti-Corin antibody. Examples of Corin-positive cells include cells expressing Corin protein on their cell surface in an amount that can be recognized by the "cell selection method" described below. Examples of the Corin-positive cells include midbrain floor plate cells, and further include cells that can be differentiated into midbrain floor plate cells and dopaminergic neural progenitor cells under specific culture conditions.

[0062] <Step of measuring the concentration of NT-3> In this step, the concentration of NT-3 is measured in the culture supernatant of the culture medium obtained by culturing the pluripotent stem cells in a medium containing an inducer of differentiation into neural cells of the midbrain floor plate region.

[0063] (NT-3 Concentration) In this embodiment, "NT-3" refers to neurotrophin-3, a type of neurotrophic factor. The method for measuring the concentration of NT-3 in the culture supernatant is not particularly limited, and examples thereof include ELISA (Enzyme-Linked Immunosorbent Assay), immunochemiluminescence (CLIA), latex agglutination, radioimmunoassay, immunoturbidimetry, enzyme activity measurement, dye binding, Western blotting, HumanMAP, mass spectrometry, immunochromatography, and methods using a multiplex suspension array system. An example of the multiplex suspension array system is Luminex (registered trademark) manufactured by Genetic Lab Co., Ltd.

[0064] In one aspect of this embodiment, the concentration of NT-3 in the culture supernatant may be measured using a measurement method with high detection sensitivity. Use of a measurement method with high detection sensitivity enables sensitive and early detection of changes in the NT-3 concentration in a low concentration range (e.g., 5 pg / ml to 20 pg / ml). Examples of such measurement methods with high detection sensitivity include digital ELISA and ProQuantum High Sensitivity Immunoassay (ProQuantum High Sensitivity Immunoassay, Thermo Fisher Scientific).

[0065] In one aspect of this embodiment, the method for measuring the NT-3 concentration can be appropriately selected depending on the cell line used, the production scale, the purpose of evaluation, etc. Furthermore, depending on the specific production process, a method for monitoring only the increase in NT-3 concentration or a method for monitoring both the increase in NT-3 concentration and the decrease in NT-3 concentration may be selected, and the change in NT-3 concentration during the production process may be measured in advance, and a method suitable for each production process may be adopted.

[0066] The culture supernatant is preferably collected from the culture medium at any time between 48 and 240 hours after the initiation of culture in a medium containing a differentiation inducer of the pluripotent stem cells (also referred to as "after the initiation of differentiation induction"). The culture supernatant is preferably collected from the culture medium at any time between 48 and 192 hours after the initiation of culture of the pluripotent stem cells. Here, "any time between 48 and 240 hours after the initiation of culture" is not particularly limited and can be arbitrarily selected, as long as NT-3 can be detected in the culture supernatant when differentiation into neural cells of the midbrain floor plate region proceeds normally. That is, the timing for collecting the culture supernatant from the culture medium can be appropriately determined depending on the method for producing the target cells, such as neural cells of the midbrain floor plate region or dopaminergic neural progenitor cells, based on whether a concentration of NT-3 equal to or greater than the reference concentration can be detected at any time between 48 and 240 hours after the initiation of culture. For example, in one aspect, the culture supernatant may be collected from the culture medium any time between 48 and 96 hours after the initiation of culturing the pluripotent stem cells. In another aspect of this embodiment, the culture supernatant may be collected from the culture medium any time between 48 and 72 hours after the initiation of culturing the pluripotent stem cells. In another aspect of this embodiment, the culture supernatant may be collected from the culture medium any time between 120 and 192 hours after the initiation of culturing the pluripotent stem cells (after the initiation of differentiation induction). The number of times the culture supernatant is collected is not particularly limited. Here, "initiating culture in a medium containing a differentiation inducer for pluripotent stem cells" refers to initiating culture of pluripotent stem cells in a medium that contains an inducer for differentiation into neural cells of the midbrain floor plate region, as described below, and / or does not contain factors necessary for maintaining pluripotency, such as bFGF, and is therefore unable to maintain pluripotency. In one aspect, this refers to initiating culture by replacing the medium used for culturing pluripotent stem cells (e.g., expansion culture) with a medium used for differentiation induction.

[0067] After differentiation induction begins, medium exchange is performed regardless of whether or not NT-3 concentration is measured in order to ensure appropriate cell culture during the differentiation induction step. The frequency of medium exchange is not particularly limited as long as the differentiation inducer and nutrient sources necessary for the cells are appropriately replenished; however, medium exchange is typically performed approximately every 12 hours, approximately every 24 hours, approximately every 48 hours, or approximately every 72 hours. Furthermore, the medium exchange method may involve replacing the entire medium, or a partial medium exchange, such as ¾, half, ⅓, or ¼. Here, when culturing is continued using a medium consisting of the same components, medium exchange is generally performed in a consistent manner throughout the duration of the culture. Therefore, the concentration of NT-3 contained in the culture supernatant is affected by the frequency and method of medium exchange. Therefore, although the detected NT-3 concentration varies depending on the individual manufacturing process, the culture supernatant can be collected at the appropriate time. Furthermore, an appropriate reference concentration can be set for each manufacturing process. In other words, when setting the reference concentration, it is desirable to synchronize it with the collection of culture supernatant performed during the manufacturing process, taking into account the medium exchange method and schedule. It is desirable that the "NT-3 concentration" correlates with the amount of NT-3 produced by cells over a certain period of time. For example, in the case of a concentration in a culture supernatant collected after a medium exchange in which the entire volume is exchanged over a certain period of time, the concentration of NT-3 secreted from the previous medium exchange to the current medium exchange is measured, and this refers to the "NT-3 concentration" herein. Therefore, when a culture supernatant is collected during a medium exchange, this corresponds to the NT-3 concentration in the culture supernatant discarded during the medium exchange procedure. For example, the "NT-3 concentration" in the waste culture supernatant from a medium exchange performed every 24 hours corresponds to the amount of NT-3 secreted by cells into the culture supernatant from the time of the previous medium exchange to the time of the next medium exchange, 24 hours after the previous medium exchange. In one aspect, when a portion of the culture supernatant is sampled from a medium that has not undergone a medium exchange, i.e., when no medium exchange is performed between two samplings, the cumulative amount of NT-3 secreted by cells contained in the culture medium is measured. Therefore, if the difference between the NT-3 concentrations of the two samples is calculated, it corresponds to the "NT-3 concentration" at the time of the second sampling.When the entire medium is replaced and the waste liquid is collected, there is no problem because the entire medium is stirred. However, when collecting a portion of the medium from the culture vessel, it is desirable to consider the possibility that the NT-3 concentration may vary depending on the distance between the cells and the sampling position of the culture supernatant, and to determine the sampling position in advance, or to gently stir the medium to make the NT-3 concentration uniform before collecting the culture supernatant.

[0068] The term "culture supernatant" refers to a liquid component of a culture medium containing cells such as pluripotent stem cells and a medium. In one aspect of this embodiment, the culture supernatant can also be understood as the culture medium from which cells have been removed. In one aspect of this embodiment, the culture supernatant can also be understood as a culture supernatant at a stage in which differentiation induction into neural cells (by an SMAD inhibitor) and ventralization (by SHH, described below) are occurring. In addition, in one aspect of this embodiment, the culture supernatant can also be understood as a culture supernatant at a stage in which differentiation induction into neural cells (by an SMAD inhibitor) and ventralization (by SHH, described below) are occurring, and the stage is between 24 hours before and 24 hours after posteriorization (by enhanced WNT signaling, described below). In another aspect of this embodiment, the culture supernatant can also be understood as a culture supernatant at a stage in which differentiation induction into neural cells (by an SMAD inhibitor) and ventralization (by SHH, described below) are occurring, and the stage is between 24 hours before and 120 hours after posteriorization (by enhanced WNT signaling, described below). In another aspect of this embodiment, the culture supernatant can also be understood as a culture supernatant at a stage where differentiation induction into neural cells (by a SMAD inhibitor) has begun and ventralization (by SHH, described below) and / or posteriorization (by enhanced WNT signaling, described below) is progressing. When performing adhesion culture or cell aggregate culture, the culture supernatant can be recovered from the culture medium using a pipette or the like without centrifugation. Furthermore, the culture supernatant discarded during medium replacement can also be recovered.

[0069] In this embodiment, a culture medium for inducing differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region contains an agent for inducing differentiation into neural cells of the midbrain floor plate region. The culture medium can be prepared using a medium used for culturing animal cells as a basal medium. Examples of basal media include Glasgow's Minimum Essential Medium (GMEM) medium, Iscove's Modified Dulbecco's Medium (IMDM) medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), and mixed media thereof. The basal medium is preferably GMEM medium. The medium may or may not contain serum. A medium that does not contain serum is sometimes called a serum-free medium. If necessary, the medium may contain one or more serum substitutes, such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The medium may also contain one or more substances, such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. A preferred medium is GMEM medium containing KSR, 2-mercaptoethanol, non-essential amino acids and pyruvate.To this medium, one or more reagents (i.e., differentiation inducers) selected from the group consisting of a BMP signaling inhibitor, a TGFβ signaling inhibitor, an SHH signaling activator, FGF8 (fibroblast growth factor 8), and a Wnt signaling activator (e.g., a GSK-3β inhibitor), as described below, are added in appropriate combination, and the culture medium can be used sequentially in an appropriate order to allow for culture.

[0070] (Inducer of Differentiation into Neural System Cells of the Midbrain Floor Plate Region) In this embodiment, the term "inducer of differentiation into neural system cells of the midbrain floor plate region" refers to a drug (substance) or a combination of drugs that induces differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region. In other words, the combination of a drug and the "timing of using a medium containing the drug" required to induce differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region is also included in the concept of "inducer of differentiation into neural system cells of the midbrain floor plate region." The inducer of differentiation into neural system cells of the midbrain floor plate region is not particularly limited, but for example, differentiation inducers used in the differentiation induction method described in Non-Patent Document 2 (Frontiers in Cell and Developmental Biology, August 2020, Volume 8, Article 729) can be used. As an example, culture conditions containing an SMAD signaling inhibitor can be used, and culture conditions containing at least one SMAD signaling inhibitor are preferred. In other words, the agent for inducing differentiation into neural cells in the midbrain floor plate region preferably contains at least one SMAD signaling inhibitor. For example, when two or more SMAD signaling inhibitors are used, culture may be performed using a medium containing all of the SMAD signaling inhibitors used, or culture may be performed by combining media containing some of the SMAD signaling inhibitors used over time. In other words, all of the SMAD signaling inhibitors used may be added to the medium at the same time, or each of the SMAD signaling inhibitors used may be added to the medium sequentially at an appropriate time.

[0071] In this embodiment, the term "SMAD signaling inhibitor" refers to a substance that inhibits signal transduction mediated by the transcription factor SMAD (Small Mothers Against Decapentaplegic). The SMAD signaling inhibitor preferably includes at least one BMP signaling inhibitor and at least one TGFβ signaling inhibitor.

[0072] In this embodiment, the term "BMP signaling inhibitor" refers to a substance that inhibits signaling mediated by the signal protein BMP (bone morphogenetic protein). Examples of the BMP signaling inhibitor include proteinaceous inhibitors such as Chordin, Noggin, and Follistatin, dorsomorphin (i.e., 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine), and derivatives thereof (P.B. Yu et al. (2007), Circulation, 116: II_60; P.B. Yu et al. (2008), Nat. Chem. Biol., 4: 33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904) and LDN-193189 (i.e., 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline), DMH1, K02288, LDN-214117, LDN-212854, ML347 (LDN193719). Dorsomorphin and LDN-193189 are commercially available and are available from Sigma-Aldrich and Stemgent, respectively. The BMP signaling inhibitor preferably includes at least one selected from the group consisting of LDN-193189, Noggin, DMH1, Chordin, Follistatin, K02288, LDN-214117, LDN-212854, ML347 (LDN193719), and Dorsomorphin.

[0073] The concentration of LDN-193189 in the culture medium is not particularly limited as long as it is a concentration that inhibits BMP, but is, for example, 1 nM to 50 μM, preferably 1 nM to 1000 nM, more preferably 50 nM to 300 nM, and more specifically, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM. When another substance is used as the BMP signaling inhibitor, a concentration that exhibits BMP signaling inhibitory activity equivalent to the concentration of LDN-193189 described above can be appropriately adopted.

[0074] In this embodiment, the term "TGFβ signaling inhibitor" refers to a substance that inhibits signaling mediated by the growth factor TGFβ (Transforming Growth Factor-β). Examples of the TGFβ signaling inhibitor include substances that inhibit binding to the ALK family receptors, or substances that inhibit phosphorylation of SMAD by the ALK family. Examples include Lefty-1 (NCBI Accession No.: mouse: NM_010094, human: NM_020997), Lefty-2, SB-431542, SB-202190 (R.K. Lindemann et al., Mol. Cancer, 2003, 2:20), SB-505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Examples of such an inhibitor include cyclosporin (ALD), ...The TGFβ signaling inhibitors include SB-431542, A-83-01, Lefty-1, Lefty-2, Galunisertib (LY2157299), SB-202190, SB-525334, SB-505124, NPC30345, Prifenidone (S-7701), GW788388, E-616452 (RepSox), SD208, TP0427736, BIBF-0775, LY3200882, Vactosertib (TEW-7197), ITD-1, SD093, SD908, LY2109761, LY364947, and LY5 It is preferable to include at least one selected from the group consisting of SB-431542, A-83-01, Lefty-1, Lefty-2, Galunisertib (LY2157299), SB-202190, SB-525334, SB-505124, NPC30345, Prifenidone (S-7701), GW788388, E-616452 (RepSox), SD208, ITD-1, SD093, SD908, LY2109761, LY364947 and LY580276, more preferably at least one selected from the group consisting of SB-431542, A-83-01, Lefty-1, Lefty-2, Galunisertib (LY2157299), SB-202190, SB-525334, SB-505124, NPC30345, Prifenidone (S-7701), GW788388, E-616452 (RepSox), SD208, ITD-1, SD093, SD908, LY2109761, LY364947 and LY580276.

[0075] In one aspect of this embodiment, the BMP signaling inhibitor comprises at least one selected from the group consisting of LDN-193189, Noggin, DMH1, Chordin, Follistatin, K02288, LDN-214117, LDN-212854, ML347, and Dorsomorphin, and the TGFβ signaling inhibitor comprises SB-431542, A-83-01, Lefty-1, Lefty-2, Galunisertib (LY2157299). , SB-202190, SB-525334, SB-505124, NPC30345, Prifenidone (S-7701), GW788388, E-616452 (RepSox), SD208, TP0427736, BIBF-0775, LY3200882, Vactosertib (TEW-7197), ITD-1, SD093, SD908, LY2109761, LY364947 and LY580276.

[0076] The concentration of A-83-01 in the culture medium is not particularly limited as long as it is a concentration that inhibits ALK5, and is, for example, 1 nM to 100 μM, preferably 100 nM to 5 μM, more preferably 300 nM to 1 μM, and more specifically, includes, but is not limited to, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, and 50 μM. When another substance is used as the TGFβ signaling inhibitor, a concentration that exhibits TGFβ signaling inhibitory activity equivalent to the above-mentioned concentration of A-83-01 can be appropriately adopted.

[0077] (SHH signaling active substance and Wnt signaling active substance) In one aspect of this embodiment, the medium preferably further comprises an SHH signaling active substance and a Wnt signaling active substance.

[0078] The term "SHH signaling active substance" refers to a substance capable of enhancing signal transduction mediated by SHH (Sonic hedgehog). Examples of SHH signaling active substances include proteins belonging to the Hedgehog family (e.g., SHH and IHH) and fragments, modified forms, and mutants thereof, SHH receptors, SHH receptor agonists, Hh-Ag1.5 (Li, X., et al., Nature Biotechnology, 23, 215-221 (2005)), Smoothened Agonist, SAG (N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane), 20a-hydroxycholesterol, Purmorphamine, and derivatives thereof (Stanton BZ, Peng LF., Mol Biosyst. 6: 44-54, 2010). The SHH signaling activator preferably comprises at least one selected from the group consisting of SHH and fragments thereof (e.g., Shh (C24II) N-Terminus, Shh (C25II) N-Terminus) and modified forms thereof, SHH receptor, SHH receptor agonist, Hh-Ag1.5, smoothened agonist, 20a-hydroxycholesterol, purmorphamine, and SAG. As an example, an SMAD signaling inhibitor and an SHH signaling activator can be used in combination. When these agents are used in combination, culture may be performed using a medium containing all of the agents to be used, or culture may be performed by combining media containing some of the agents to be used over time. In other words, all of the agents to be used may be added to the medium at the same time, or each of the agents to be used may be added to the medium sequentially at an appropriate time.

[0079] The concentration of Purmorphamine in the culture medium is not particularly limited as long as it is a concentration that activates Gli2, and is, for example, 1 nM to 50 μM, preferably 100 nM to 10 μM, more preferably 500 nM to 5 μM, and more specifically, but not limited to, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, or 50 μM. When another substance is used as the SHH signaling active substance, a concentration that exhibits SHH signaling activity equivalent to the above-mentioned Purmorphamine concentration can be appropriately adopted.

[0080] The term "Wnt signaling active substance" refers to a substance that activates signal transduction caused by binding of a Wnt protein to a Frizzled receptor. The Wnt signaling active substance preferably includes at least one substance selected from the group consisting of WNT3A and a GSK-3β inhibitor.

[0081] The term "GSK-3β inhibitor" refers to a substance that inhibits the kinase activity of GSK-3β protein (for example, the ability to phosphorylate β-catenin). Examples of the GSK-3β inhibitor include BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin 3'-oxime), which is an indirubin derivative; SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), which is a maleimide derivative; GSK-3β inhibitor VII (4-dibromoacetophenone), which is a phenyl α-bromomethyl ketone compound; and L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQpSP-NH), which is a cell membrane-permeable phosphorylated peptide. ２(SEQ ID NO: 1)) and highly selective CHIR99021 (6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazolo-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile), CHIR98014, and SKL2001. These compounds are commercially available from, for example, Calbiochem and Biomol, and can be easily used, but they may also be obtained from other sources or may be prepared in-house. The GSK-3β inhibitor preferably includes at least one selected from the group consisting of CHIR99021, BIO, CHIR98014, SKL2001, SB216763, GSK-3β inhibitor VII (4-dibromoacetophenone), and L803-mts.

[0082] The concentration of CHIR99021 in the culture medium is, for example, 1 nM to 50 μM, preferably 10 nM to 20 μM, more preferably 100 nM to 10 μM, and more specifically, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, or 50 μM, but is not limited to these. When another substance is used as the Wnt signaling activator or GSK3β inhibitor, a concentration that exhibits Wnt signaling activity or GSK3β inhibitory activity equivalent to the above-mentioned CHIR99021 concentration can be appropriately adopted. As an example, a Wnt signaling activator or a GSK3β inhibitor can be used in combination with an SMAD signaling inhibitor and an SHH signaling activator. In this case, it is not necessary to add all of these agents to the medium at the same time; they may be combined and cultured over time as appropriate to enable differentiation induction. In other words, each of the agents used may be added to the medium sequentially at an appropriate time. As an example, there is a method in which the types of agents contained in the medium are increased or decreased over time, such as (1) a step of culturing in a medium containing one or more, preferably two or more, SMAD signaling inhibitors, (2) a step of culturing in a medium further containing an SHH signaling activator, and then (3) a step of culturing in a medium further containing a Wnt signaling activator.

[0083] In one aspect of this embodiment, the medium preferably contains FGF8 (fibroblast growth factor 8). The FGF8 is not particularly limited, but in the case of human FGF8, four splicing forms, FGF8a, FGF8b, FGF8e, and FGF8f, are exemplified, with FGF8b being more preferred. FGF8 is commercially available from, for example, Wako and R&D Systems and can be easily used, but it may also be obtained by forced expression in cells using methods known to those skilled in the art. As an example, FGF8 can be used in combination with an SMAD signaling inhibitor, an SHH signaling activator, and a Wnt signaling activator or a GSK3β inhibitor. In this case, it is not necessary to add all of these agents to the medium at the same time; they may be combined and cultured over time as needed to induce differentiation. In other words, each of the agents used may be added to the medium sequentially at an appropriate time.

[0084] The concentration of FGF8 in the culture medium is, for example, 1 ng / mL to 5000 ng / mL, preferably 10 ng / mL to 1000 ng / mL, and more specifically, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, or 5000 ng / mL, but is not limited to these.

[0085] <Step of Comparing the Measured NT-3 Concentration with a Reference Concentration> In this step, the measured NT-3 concentration is compared with a reference concentration. The "reference concentration" refers to the NT-3 concentration (cutoff value) that serves as a reference for determining whether the target pluripotent stem cells can differentiate into midbrain floor plate region neural cells, and can be set arbitrarily. For example, any value, including the detection limit, can be used as the reference concentration as long as it is statistically distinguishable from the detection limit. The reference concentration may be the NT-3 concentration obtained when pluripotent stem cells are cultured under specified conditions and successfully differentiated into midbrain floor plate region neural cells, or it may be a preset concentration. The preset concentration can be set, for example, as follows. That is, first, pluripotent stem cells are cultured in a medium and under culture conditions that allow differentiation induction, i.e., according to the production procedure (protocol) for midbrain floor plate region neural cells, and the NT-3 concentration in the culture supernatant is measured when target cells that achieve the goal are obtained, i.e., when the differentiation induction efficiency is good. Such culturing and measurement can be performed multiple times (at least twice, preferably at least five times), and the average NT-3 concentration measured in each step can be set as the reference concentration. Alternatively, the reference concentration can be set to a concentration that provides the sensitivity and specificity required by the user, based on the relationship between the reference concentration and the sensitivity and specificity estimated from an ROC curve derived from the NT-3 concentration in the culture supernatant when target cells that achieve the target are obtained, and the NT-3 concentration in the culture supernatant when target cells that achieve the target are not obtained, i.e., when the efficiency of differentiation induction is poor, when pluripotent stem cells are cultured in a medium and under culture conditions that enable differentiation.

[0086] Here, in this specification, "when target cells that achieve the goal are obtained" or "when differentiation induction efficiency is good" refers to when target cells are obtained in an amount (proportion) or more that meets the goal, i.e., production results. The "amount (proportion) that meets the goal" may be set appropriately, and examples include 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the total number of cells being cultured.

[0087] As used herein, "when target cells that achieve the target are not obtained" or "when the efficiency of differentiation induction is poor" refers to a case where target cells are not obtained in a quantity (proportion) that matches the target despite differentiation induction being performed according to the manufacturing instructions (protocol) for neural cells of the midbrain floor plate region, which is a cell population of dopaminergic neural progenitor cells or their intermediate, midbrain floor plate cells. It is desirable to obtain "incompatible" results, in which target cells are not obtained in a quantity that matches the target, at least twice, and preferably at least five times, and then set the average NT-3 concentration measured in each case as the "NT-3 concentration in the culture supernatant when differentiation induction efficiency is poor." Alternatively, "when target cells that achieve the target are not obtained" or "when differentiation induction efficiency is poor" may be, for example, a case where culture is performed in a medium or culture conditions that intentionally result in poor differentiation induction efficiency in order to set a reference value, i.e., a model case of "incompatible" in which target cells are not obtained in a quantity that matches the target. A model case of such incompatibility is differentiation induction under conditions that intentionally deviate from the specifications of the manufacturing protocol for neural cells of the midbrain floor plate region, which are a cell population of dopaminergic neural progenitor cells or their intermediate, midbrain floor plate cells, but that are required to be appropriately controlled in the manufacturing protocol. In this case, it is desirable to obtain "incompatibility" results, in which a target amount of target cells is not obtained, at least two times, preferably at least five times, by differentiation induction that deviates from the specifications, and then set the average NT-3 concentration measured in each case as the "NT-3 concentration in the culture supernatant when differentiation induction efficiency is poor." Here, "conditions that deviate from the specifications of the manufacturing protocol" refer to conditions under which differentiation induction into target cells proceeds appropriately but a target amount is not achieved. Conditions that do not induce differentiation into target cells or conditions that significantly affect differentiation into target cells are not appropriate as model cases for the purpose of setting standard values, and such conditions are not included in "conditions that deviate from the provisions of some manufacturing procedures" for the purpose of determining standard values.For example, it is desirable that culture conditions under which the proportion of target cells in the total cells being cultured is always 15% or less, 10% or less, or 5% or less are not included in the above-mentioned "conditions that deviate from the provisions of the partial manufacturing procedure manual." For example, depending on the proportion of target cells in the total cells set as the above-mentioned "amount (proportion) that meets the target," it is also desirable that culture conditions under which the proportion is 20% or less (15% or less, 10% or less, or 5% or less) of that proportion are not included in the above-mentioned "conditions that deviate from the provisions of the partial manufacturing procedure manual."

[0088] Here, conditions that need to be properly managed in the manufacturing procedure manual (protocol) include culture conditions that may affect the production efficiency of the target cells, assuming that basic differentiation induction methods are maintained. Examples include culture conditions such as medium components, freshness of the medium, medium temperature, medium pH, and carbon dioxide concentration, handling of cells with minimal physical stress (techniques such as pipetting and separation), avoiding contamination with bacteria or viruses, and the method and frequency of passaging and medium change.

[0089] Furthermore, the appropriate timing for detecting NT-3 concentration can be appropriately set by culturing pluripotent stem cells in a medium and culture conditions that allow differentiation induction, i.e., culturing them according to the manufacturing procedures (protocols) for neural cells in the midbrain floor plate region or dopaminergic neural progenitor cells, so that the NT-3 concentration can be effectively measured. For example, culture and measurement can be performed multiple times (e.g., twice, preferably five times) over time, and the timing at which the NT-3 concentration can be detected can be appropriately set. In one embodiment, a portion of the waste liquid can be collected when the medium is replaced, and the NT-3 concentration can be measured, and an appropriate measurement frequency can be set accordingly. For example, measurement can be performed every time the medium is replaced.

[0090] Specifically, the reference concentration is preferably set at a single point between 0.1 pg / ml and 10,000 pg / ml, and more preferably set at a single point between 0.3 pg / ml and 3,000 pg / ml, more specifically, between 0.3 pg / ml and 100 pg / ml.

[0091] In one aspect of this embodiment, the reference concentration may be the concentration of NT-3 before the amount of NT-3 secretion increases. For example, the reference concentration may be the concentration of NT-3 measured within 48 hours, preferably within 24 hours, after the start of differentiation induction.

[0092] <Step of Determining That Cells in a Culture Medium Are Capable of Differentiating into Midbrain Floor Plate Region Neural Cells> In this step, when the NT-3 concentration is equal to or greater than the reference concentration, the cells (e.g., pluripotent stem cells, cells derived from the pluripotent stem cells) in the culture medium at the time the NT-3 concentration was measured are determined to be capable of differentiating into the midbrain floor plate region neural cells, i.e., capable of differentiating into midbrain floor plate region neural cells, and further capable of differentiating into dopaminergic neural progenitor cells. In one aspect of this embodiment, "capable of differentiating" can also be understood as "possibly capable of differentiating." Furthermore, in one aspect of this embodiment, "capable of differentiating" may also mean that, in the production of midbrain floor plate region neural cells, a target amount of midbrain floor plate region neural cells can be expected to be obtained in the differentiation induction step. Furthermore, in one aspect of this embodiment, "determining that cells are capable of differentiating" refers to determining that the differentiation induction step is progressing smoothly in the production of midbrain floor plate region neural cells. In one aspect of this embodiment, in the production of neural cells of the midbrain floor plate region, a case in which it is estimated that the target amount of neural cells of the midbrain floor plate region cannot be obtained in the differentiation induction step corresponds to a case in which "it cannot be determined that they can be differentiated." Furthermore, the "NT-3 concentration" of the present invention can be used not only to determine whether cells in a culture medium can differentiate into neural cells of the midbrain floor plate region, but also to determine at an early stage of the production process whether the cells satisfy the required quality and quantity as intermediate cells for producing "dopamine neural progenitor cells, which are drug substances of a level (grade) that can be used for treatment as a cellular pharmaceutical."

[0093] The method of the present invention makes it possible to determine early on whether cells are properly destined to differentiate into target cells at an early stage of inducing differentiation of pluripotent stem cells into dopaminergic neural progenitor cells. Furthermore, the method of the present invention is extremely useful for managing the manufacturing process. Furthermore, the method of the present invention can also be used to determine whether a pluripotent stem cell line is suitable for inducing differentiation into target cells. Furthermore, the method of the present invention can be used to determine the efficiency of differentiation induction when optimizing the conditions for scaling up manufacturing.

[0094] In one aspect of this embodiment, the phrase "the concentration of NT-3 is equal to or greater than the reference concentration" encompasses cases where the concentration of NT-3 is equal to the reference concentration and cases where the concentration of NT-3 is higher than the reference concentration. For example, when the concentration of NT-3 is 1 to 1000 times the reference concentration, it is preferable to determine that the cells in the culture medium are capable of differentiating into neural cells of the midbrain floor plate region. When the concentration of NT-3 is 1.2 to 1000 times the reference concentration, it is more preferable to determine that the cells contained in the culture medium at the time the concentration of NT-3 was measured are capable of differentiating into neural cells of the midbrain floor plate region.

[0095] The percentage of a cell population determined by the determination method of the present invention to be capable of differentiating into neural cells of the midbrain floor plate region that will differentiate into neural cells of the midbrain floor plate region may vary depending on the culture conditions and the pluripotent stem cells used, but for example, it is thought that 20% or more, preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the total cells being cultured will differentiate into neural cells of the midbrain floor plate region.

[0096] In one aspect of this embodiment, from the viewpoint of efficient differentiation induction, when the NT-3 concentration is less than the reference concentration, the cells in the culture medium or the culture medium itself may be discarded. The decision on whether to discard and the timing of discarding are desirably changed as appropriate depending on the production method, the scale of the culture, the type and strain of cells used, the final amount of cells required, the target grade, whether preliminary culture is possible and its progress, the overall production schedule, the presence or absence of other determination methods, the number of days required to make the decision, costs, etc.

[0097] <<Method (2) of Determining the Differentiation Potential of Cells in a Culture Solution During Differentiation of Pluripotent Stem Cells into Neural System Cells of the Midbrain Floor Plate Region>> A second method of determining the differentiation potential of cells in a culture solution during differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region according to this embodiment is a method of determining the differentiation potential of cells in a culture solution during differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region, comprising the steps of: measuring the concentrations of NT-3 in a first culture supernatant and a second culture supernatant in a culture solution obtained by culturing the pluripotent stem cells in a medium containing an inducer for differentiation into neural system cells of the midbrain floor plate region; determining the rate of change in the NT-3 concentration from the NT-3 concentration in the first culture supernatant and the NT-3 concentration in the second culture supernatant; comparing the absolute value of the rate of change in the NT-3 concentration with a reference rate of change; and determining that the cells in the culture medium are capable of differentiating into neural cells of the midbrain floor plate region when the absolute value of the rate of change in the NT-3 concentration is equal to or greater than the reference rate of change, wherein the first culture supernatant is collected from the culture medium any time between 48 and 192 hours after the start of culturing the pluripotent stem cells, and the second culture supernatant is collected from the culture medium any time between 24 and 96 hours before or 24 and 96 hours after the collection of the first culture supernatant. These methods are described in detail below. The pluripotent stem cells can be any of the cells listed in the first method described above.

[0098] <Step of Measuring the Concentration of NT-3> In this step, the concentrations of NT-3 are measured in the first culture supernatant and the second culture supernatant in the culture medium obtained by culturing the pluripotent stem cells in a medium containing an inducer of differentiation into neural cells of the midbrain floor plate region. The method for measuring the concentration can be any of the methods listed in the first method described above.

[0099] The first culture supernatant is a culture supernatant collected from the culture medium any time between 48 and 192 hours after the initiation of culturing the pluripotent stem cells, and in one aspect may be a culture supernatant collected from the culture medium any time between 48 and 96 hours after the initiation of culturing the pluripotent stem cells. In another aspect, the first culture supernatant may be a culture supernatant collected from the culture medium any time between 48 and 72 hours after the initiation of culturing the pluripotent stem cells.

[0100] In one embodiment, the second culture supernatant is collected from the culture medium at or after the initiation of differentiation and before the collection of the first culture supernatant, for example, 24 to 96 hours (preferably 24 to 72 hours) after the initiation of differentiation. The second culture supernatant is collected from the culture medium before the collection of the first culture supernatant. That is, the second culture supernatant can be collected from the culture medium after the collection of the first culture supernatant. That is, the second culture supernatant can be collected from the culture medium after the collection of the first culture supernatant. That is, the second culture supernatant can be collected from the culture medium after the collection of the first culture supernatant. That is, the second culture supernatant can be collected from the culture medium after the collection of the first culture supernatant. The second culture supernatant can be collected from the culture medium after the collection of the first culture supernatant. The second culture supernatant can be collected from the culture medium after the collection of the first culture supernatant. The second culture supernatant can be collected from the culture medium after the collection of the first culture supernatant. The second culture supernatant can be collected from the culture medium after the collection of the first culture supernatant. Here, "completion of differentiation into neural cells of the midbrain floor plate region" refers to a state in which cell differentiation has progressed to the point where markers of neural cells of the midbrain floor plate region are expressed. In this case, the culture medium may contain neural cells that have further differentiated from neural cells of the midbrain floor plate region. Specifically, this includes differentiation stages in which multiple markers of neural cells in the midbrain floor plate region are detected. Examples of such markers include Corin, Foxa2, Lmx1a, Lmx1b, and CD142. The second culture supernatant is preferably collected from the culture medium at any time between 24 and 48 hours before collection of the first culture supernatant or at any time between 24 and 96 hours (more preferably, 24 and 72 hours) after collection of the first culture supernatant. Here, "any time between 24 and 96 hours before collection of the first culture supernatant" refers to any time in the period between 24 hours before collection of the first culture supernatant and 96 hours before collection of the first culture supernatant.

[0101] The composition of the medium for culturing the pluripotent stem cells can be the same as that of the first method described above.

[0102] <Step of determining the rate of change in NT-3 concentration> In this step, the rate of change in NT-3 concentration is determined from the concentration of NT-3 in the first culture supernatant and the concentration of NT-3 in the second culture supernatant. The rate of change in NT-3 concentration can be determined by the following formula: When the second culture supernatant is collected before the first culture supernatant: (rate of change in NT-3 concentration) = {(NT-3 concentration in the first culture supernatant) - (NT-3 concentration in the second culture supernatant)} / {(time when the first culture supernatant is collected) - (time when the second culture supernatant is collected)} When the second culture supernatant is collected after the first culture supernatant: (rate of change in NT-3 concentration) = {(NT-3 concentration in the second culture supernatant) - (NT-3 concentration in the first culture supernatant)} / {(time when the second culture supernatant is collected) - (time when the first culture supernatant is collected)}

[0103] In one aspect of this embodiment, the NT-3 concentration in the culture supernatant may temporarily increase and decrease over 24 or 48 hours during the period from the initiation of differentiation to day 12 (the period from the initiation of culture of the pluripotent stem cells to 288 hours after the initiation of culture of the pluripotent stem cells). In another aspect of this embodiment, the NT-3 concentration in the culture supernatant may temporarily increase during the period from the initiation of differentiation to day 12, with the increased period lasting approximately 168 hours. Here, the "elevated period" refers to the period during which the NT-3 concentration is higher than the baseline value. As long as the NT-3 concentration is higher than the baseline value, it is included in the "elevated period" even if it remains constant or decreases temporarily. In one aspect of this embodiment, the NT-3 concentration in the culture supernatant may continuously increase during the period from the initiation of differentiation to day 12 (the period from the initiation of culture of the pluripotent stem cells to 288 hours after the initiation of culture of the pluripotent stem cells). The timing of collecting the first culture supernatant and the second culture supernatant can be appropriately set depending on the characteristics of the change in the NT-3 concentration in each differentiation induction step.

[0104] <Step of Comparing the Absolute Value of the Rate of Change in NT-3 Concentration with a Reference Rate of Change> In this step, the absolute value of the rate of change in NT-3 concentration is compared with a reference rate of change. The "reference rate of change" is an absolute value (cutoff value) of the rate of change in NT-3 concentration that serves as a standard for determining whether a target pluripotent stem cell can differentiate into midbrain floor plate region neural cells, and can be set arbitrarily. The reference rate of change may be the absolute value of the rate of change in NT-3 concentration determined when pluripotent stem cells known to differentiate into midbrain floor plate region neural cells are cultured under specified conditions, or it may be a preset absolute value of a rate of change. The preset absolute value of the rate of change can be set, for example, as follows. That is, first, pluripotent stem cells are cultured in a medium and culture conditions capable of inducing differentiation, i.e., in accordance with the production procedure (protocol) for midbrain floor plate region neural cells, and the "absolute value of the rate of change in NT-3 concentration" is determined when target cells that achieve the goal are obtained, i.e., when the differentiation induction efficiency is good. Such culture and measurement can be performed multiple times (at least two times, preferably at least five times), and the average of the "absolute value of the rate of change of NT-3 concentration" determined in each case can be set as the reference rate of change. Alternatively, when pluripotent stem cells are cultured in a medium and culture conditions capable of inducing differentiation, the absolute value of the rate of change of NT-3 concentration when target cells achieving the target are obtained can be used to estimate the relationship between the reference rate of change and the sensitivity and specificity estimated from an ROC curve derived from the "absolute value of the rate of change of NT-3 concentration" when target cells achieving the target are not obtained, i.e., when the efficiency of differentiation induction is poor. Here, the meanings of "when target cells achieving the target are obtained" and "when differentiation induction efficiency is good," as well as "when target cells achieving the target are not obtained" and "when differentiation induction efficiency is poor," are the same as those described in the section "Step of comparing the measured NT-3 concentration with the reference concentration" in the first method.

[0105] Furthermore, in one aspect of this embodiment, when the second culture supernatant is collected before the first culture supernatant, the timing of collection of each culture supernatant may be set so that it is possible to detect that the NT-3 concentration in the first culture supernatant is higher than the NT-3 concentration in the second culture supernatant. In this case, the above-mentioned rate of change may be calculated, or the NT-3 concentration in the first culture supernatant may be determined to be higher than the NT-3 concentration in the second culture supernatant. Furthermore, in one aspect of this embodiment, when the second culture supernatant is collected after the first culture supernatant, the timing of collection of each culture supernatant may be set so that it is possible to detect that the NT-3 concentration in the second culture supernatant is lower than the NT-3 concentration in the first culture supernatant. In this case, the above-mentioned rate of change may be calculated, or the NT-3 concentration in the second culture supernatant may be determined to be lower than the NT-3 concentration in the first culture supernatant.

[0106] The reference rate of change is preferably set to a single point between 0.1 pg / ml·day and 500 pg / ml·day, and more preferably set to a single point between 5 pg / ml·day and 50 pg / ml·day.

[0107] <Step of determining whether cells in the culture medium can differentiate into neural cells of the midbrain floor plate region> In this step, when the absolute value of the rate of change in the concentration of NT-3 is equal to or greater than the reference rate of change, it is determined that the cells in the culture medium can differentiate into neural cells of the midbrain floor plate region.

[0108] In one aspect of this embodiment, the phrase "the absolute value of the rate of change in NT-3 concentration is equal to or greater than the reference rate of change" encompasses cases where the absolute value of the rate of change in NT-3 concentration is equal to the reference rate of change, and cases where the absolute value of the rate of change in NT-3 concentration is higher than the reference rate of change. For example, it is preferable to determine that the cells in the culture medium are capable of differentiating into neural cells of the midbrain floor plate region when the absolute value of the rate of change in NT-3 concentration is 1 to 10,000 times the reference rate of change, and it is more preferable to determine that the cells in the culture medium are capable of differentiating into neural cells of the midbrain floor plate region when the absolute value of the rate of change in NT-3 concentration is 1 to 1,000 times the reference rate of change.

[0109] The percentage of a cell population determined to be capable of differentiating into neural cells of the midbrain floor plate region that differentiates into neural cells of the midbrain floor plate region may vary depending on the culture conditions and the pluripotent stem cells used, but it is thought that, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the total cells being cultured will differentiate into neural cells of the midbrain floor plate region.

[0110] In one aspect of this embodiment, from the viewpoint of efficient differentiation induction, when the absolute value of the rate of change in the NT-3 concentration is less than the reference rate of change, the cells in the culture medium or the culture medium itself may be discarded. The decision on whether to discard and the timing of discarding are desirably changed as appropriate depending on the production method, the scale of the culture, the type and strain of cells used, the final amount of cells required, the target grade, whether preliminary culture is possible and its progress, the overall production schedule, the presence or absence of other determination methods, the number of days required to make the decision, costs, etc.

[0111] <<Method (3) for Determining the Differentiation Potential of Cells in a Culture Solution During Differentiation of Pluripotent Stem Cells into Neural System Cells of the Midbrain Floor Plate Region>> A third method for determining the differentiation potential of cells in a culture solution during differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region according to this embodiment is a method for determining the differentiation potential of cells in a culture solution during differentiation of pluripotent stem cells into neural system cells of the midbrain floor plate region, comprising the steps of: measuring the concentrations of NT-3 in a first culture supernatant, a second culture supernatant, and a third culture supernatant in a culture solution obtained by culturing the pluripotent stem cells in a medium containing an inducer for differentiation into neural system cells of the midbrain floor plate region; comparing the concentrations of NT-3 in the first culture supernatant, the second culture supernatant, and the third culture supernatant; determining that the cells in the culture medium are capable of differentiating into neural cells of the midbrain floor plate region when the concentration of NT-3 in the first culture supernatant is higher than the concentrations of NT-3 in the second culture supernatant and the third culture supernatant, wherein the first culture supernatant is collected from the culture medium any time between 48 hours and 192 hours after the start of culturing the pluripotent stem cells, the second culture supernatant is collected from the culture medium any time between 24 hours and 96 hours before the collection of the first culture supernatant, and the third culture supernatant is collected from the culture medium any time between 24 hours and 96 hours after the collection of the first culture supernatant.

[0112] <Step of Measuring the Concentration of NT-3> In this step, the concentrations of NT-3 are measured in the first culture supernatant, the second culture supernatant, and the third culture supernatant in the culture medium obtained by culturing the pluripotent stem cells in a medium containing an inducer of differentiation into neural cells of the midbrain floor plate region. The method for measuring the concentration can be any of the methods listed in the first method described above.

[0113] The first culture supernatant may be a culture supernatant collected from the culture medium any time between 48 and 192 hours after the initiation of culturing the pluripotent stem cells, or may be a culture supernatant collected from the culture medium any time between 48 and 96 hours after the initiation of culturing the pluripotent stem cells. More preferably, the first culture supernatant is a culture supernatant collected from the culture medium any time between 48 and 72 hours after the initiation of culturing the pluripotent stem cells.

[0114] In one embodiment, the second culture supernatant is collected from the culture medium at or after the initiation of differentiation and before the collection of the first culture supernatant, for example, 24 to 96 hours (preferably 24 to 72 hours) after the initiation of differentiation, but before the collection of the first culture supernatant. That is, the second culture supernatant is collected from the culture medium at any time between 24 and 96 hours (more preferably, 24 to 72 hours) before the collection of the first culture supernatant, and is preferably collected from the culture medium at any time between 24 and 48 hours before the collection of the first culture supernatant. Here, in one aspect of this embodiment, the timing of collection of each culture supernatant may be set so that it is possible to detect that the concentration of NT-3 in the second culture supernatant is higher than the concentration of NT-3 in the first culture supernatant.

[0115] The third culture supernatant is a culture supernatant collected from the culture medium at any time from 24 hours after collection of the first culture supernatant until the completion of differentiation into neural cells of the midbrain floor plate region. The third culture supernatant is preferably collected from the culture medium at any time between 24 hours and 96 hours after collection of the first culture supernatant, and is preferably collected from the culture medium at any time between 24 hours and 72 hours after collection of the first culture supernatant. Furthermore, in one aspect of this embodiment, the timing of collection of each culture supernatant may be set so that the NT-3 concentration in the third culture supernatant is detected to be lower than the NT-3 concentration in the first culture supernatant. Here, "completion of differentiation into neural cells of the midbrain floor plate region" refers to a state in which cell differentiation has progressed to the point where markers for neural cells of the midbrain floor plate region are expressed. In this case, the culture medium may contain neural cells that have further differentiated from neural cells of the midbrain floor plate region. Specifically, this includes differentiation stages in which multiple markers of neural cells in the midbrain floor plate region are detected, such as Corin, Lrtm1, Foxa2, Lmx1a, Lmx1b, and CD142.

[0116] The composition of the medium for culturing the pluripotent stem cells can be the same as that of the first method described above.

[0117] <Step of Comparing NT-3 Concentrations> In this step, the concentrations of NT-3 in the first culture supernatant, the second culture supernatant, and the third culture supernatant are compared.

[0118] <Step of determining whether cells in the culture medium can differentiate into neural cells of the midbrain floor plate region> In this step, when the concentration of NT-3 in the first culture supernatant is higher than the concentration of NT-3 in the second culture supernatant and the concentration of NT-3 in the third culture supernatant, it is determined that the cells in the culture medium can differentiate into neural cells of the midbrain floor plate region.

[0119] In one aspect of this embodiment, when the difference in concentration between the NT-3 concentration in the first culture supernatant and the lower of the NT-3 concentrations in the second culture supernatant and the third culture supernatant is 5 pg / ml or more, it is preferable to determine that the cells in the culture medium are capable of differentiating into neural cells of the midbrain floor plate region, and when the difference in concentration is 10 pg / ml or more, it is more preferable to determine that the cells in the culture medium are capable of differentiating into neural cells of the midbrain floor plate region.

[0120] The percentage of a cell population determined to be capable of differentiating into neural cells of the midbrain floor plate region that differentiates into neural cells of the midbrain floor plate region may vary depending on the culture conditions and the pluripotent stem cells used, but it is thought that, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the total cells being cultured will differentiate into neural cells of the midbrain floor plate region.

[0121] In one aspect of this embodiment, from the viewpoint of efficient differentiation induction, when the concentration of NT-3 in the first culture supernatant is the same as or lower than the concentrations of NT-3 in the second culture supernatant and the third culture supernatant, the cells in the culture medium or the culture medium itself may be discarded. The decision on whether to discard and the timing of discarding are desirably changed as appropriate depending on the production method, the scale of the culture, the type and strain of cells used, the final amount of cells required, the target grade, whether preliminary culture is possible and its progress, the overall production schedule, the presence or absence of other determination methods, the number of days required to make the decision, costs, etc.

[0122] <Method of assessment using multiple factors> In one embodiment, the method of assessment of the present invention can be carried out by combining, in addition to the above-mentioned NT-3, one or more substances other than the above-mentioned NT-3 that are detected in the culture supernatant during the process of differentiation into neural cells in the midbrain floor plate region as an indicator for assessment.

[0123] The "one or more substances other than NT-3" is not particularly limited, and can be appropriately selected from substances whose concentration in the culture supernatant is found to be correlated with differentiation into neural cells in the midbrain floor plate region, and which have been determined to be useful for the assessment method of the present invention by, for example, univariate logistic regression analysis.

[0124] When the determination method of the present invention is carried out by combining the above-mentioned NT-3 with "one or more substances other than NT-3," it is possible to first use multivariate logistic regression analysis, Cox regression analysis, or the like to determine an optimal model formula for determining the differentiation ability of the midbrain floor plate region into nervous system cells in the above-mentioned combination, and then determine the differentiation ability of the midbrain floor plate region into nervous system cells based on the model formula thus determined.

[0125] <Determination Method Using a Culture Device> In one aspect of this embodiment, the determination method can be used in a culture device for inducing differentiation of pluripotent stem cells. Examples of the culture device include a culture device equipped with a culture vessel for culturing cells of interest in liquid medium, a medium supply unit for supplying fresh liquid medium to the culture vessel, and a medium discharge unit for discharging the liquid medium from the culture vessel. The liquid medium collected from the medium discharge unit for medium replacement corresponds to the culture supernatant in the methods (1) to (3) for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region. Examples of such a culture device include the "iACE2" automated mass culture device for iPS cells manufactured by Hitachi, Ltd. The culture device may also include a culture vessel for culturing target cells in liquid medium, a culture medium supply unit for supplying fresh liquid medium to the culture vessel, a culture medium discharge unit for discharging the liquid medium from the culture vessel, a measurement unit for measuring the concentration of NT-3 contained in the discharged liquid medium, and a differentiation potential assessment unit for determining whether the target cells can differentiate into neural cells of the midbrain floor plate region based on the measured NT-3 concentration. The culture device may also include a system for automatically changing the medium during cell culture, allowing the medium to be discharged and new medium to be injected. Therefore, the assessment method of the present invention can be carried out by non-invasively collecting the discharged medium, i.e., waste liquid, and subjecting it to component analysis. For example, an analyzer (measurement unit) for measuring NT-3 contained in the waste liquid collected during medium exchange can be incorporated into the culture device, and only lots with the ability to differentiate into neural cells of the midbrain floor plate region can be selected for continued culture.

[0126] In order to enable the seeding, cultivation, and observation of cells to be carried out in a sterile environment, it is preferable that the culturing device uses a completely closed flow path module for the culture vessel and the medium flow path.

[0127] The above describes a method for assessing the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region according to the present embodiment. This assessment method is useful in established methods for producing dopaminergic neural progenitor cells, which are obtained by inducing differentiation from pluripotent stem cells and serve as active ingredients in cellular pharmaceuticals. When implementing this assessment method, it is desirable to first perform a method for producing dopaminergic neural progenitor cells to understand the NT-3 secretion pattern, and then determine the optimal timing for measuring the NT-3 secretion level for each production method. This assessment method can be suitably applied at least to production methods capable of producing cells of the midbrain floor plate region (also referred to as midbrain floor plate cells), i.e., Corin-positive cells, capable of producing a certain number of Corin-positive cells or more.

[0128] The above-mentioned determination method is preferably used in the step of inducing differentiation of iPS cells that have been cultured to maintain undifferentiation by a method well known to those skilled in the art. That is, as a culture method for maintaining the pluripotency of iPS cells, the determination method can be suitably applied as long as usable iPS cells cultured under appropriate culture conditions are used.

[0129] <<Method for producing dopaminergic neural progenitor cells or their precursor cells>> The method for producing dopaminergic neural progenitor cells or their precursor cells according to this embodiment is a method for producing dopaminergic neural progenitor cells or their precursor cells from pluripotent stem cells, comprising: (A) a step of initiating culture of the pluripotent stem cells under culture conditions that allow differentiation into neural system cells of the midbrain floor plate region; (B) a step of performing a method for determining the differentiation potential of cells in a culture medium in the differentiation of the pluripotent stem cells into neural system cells of the midbrain floor plate region between 0 and 288 hours after the initiation of culture of the pluripotent stem cells; and (C) a step of deciding to further continue culturing the cells in the culture medium that have been determined in step (B) to be capable of differentiating into neural system cells of the midbrain floor plate region, and differentiating the cells in the culture medium into neural system cells of the midbrain floor plate region. (D) culturing the neural cells in the midbrain floor plate region under culture conditions that allow differentiation into the dopaminergic neural progenitor cells, thereby differentiating the cells into the dopaminergic neural progenitor cells or their precursor cells.

[0130] <Step (A): Initiating the culture of pluripotent stem cells> In step (A), the culture of the pluripotent stem cells is initiated under culture conditions that allow the differentiation of the pluripotent stem cells into neural cells of the midbrain floor plate region. The cells described above can be used as the pluripotent stem cells.

[0131] (Culture conditions enabling differentiation into neural cells of the midbrain floor plate region) Culture conditions (medium, temperature, carbon dioxide concentration, etc.) enabling differentiation into neural cells of the midbrain floor plate region are described below. The composition of the medium for culturing the pluripotent stem cells can be the same as that described for the first method.

[0132] The temperature at which pluripotent stem cells are cultured is preferably 30°C or higher and 40°C or lower, and more preferably 36°C or higher and 38°C or lower.

[0133] The carbon dioxide concentration during the culture of pluripotent stem cells is preferably 2% or more and 5% or less, and more preferably 4% or more and 5% or less.

[0134] The cell density when culturing pluripotent stem cells is 10 cells / cm ２ More than 100 million cells / cm ２ Preferably, the number of cells per cm is 100 or less. ２ More than 1 million cells / cm ２ More preferably, it is:

[0135] In one aspect of this embodiment, step (A) preferably involves adhesion culture of pluripotent stem cells on an extracellular matrix. "Adhesion culture on an extracellular matrix" refers to culturing using a culture vessel coated with an extracellular matrix. The coating treatment can be performed by placing a solution containing the extracellular matrix in the culture vessel and then appropriately removing the solution.

[0136] <Step (B): Performing a method for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region> In step (B), a method for determining the differentiation potential of cells in a culture medium during differentiation of the pluripotent stem cells into neural cells of the midbrain floor plate region is performed between 0 and 288 hours after the start of culturing the pluripotent stem cells. In one aspect of this embodiment, step (B) can also be understood as a step of completing the determination by the method for determining the differentiation potential of cells in a culture medium during differentiation of the pluripotent stem cells into neural cells of the midbrain floor plate region between 0 and 288 hours after the start of culturing the pluripotent stem cells. In one aspect of this embodiment, "between 0 hours and 288 hours after the start of culturing the pluripotent stem cells" can be understood as "the period until neural cells in the midbrain floor plate region are generated from the pluripotent stem cells," or "the period until at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the total number of neural cells in the midbrain floor plate region are generated from the pluripotent stem cells."

[0137] Step (B) may be performed using any of the above-described methods 1, 2, and 3. Note that the culture of cells (pluripotent stem cells, cells derived from the pluripotent stem cells, etc.) in the culture medium is continued during step (B).

[0138] <Step (C): Differentiating Cells in Culture Solution into Neural System Cells of the Midbrain Floor Plate Region> In step (C), it is decided to further continue culturing the cells in the culture solution that have been determined in step (B) to be capable of differentiating into neural system cells of the midbrain floor plate region, and the cells in the culture solution are differentiated into neural system cells of the midbrain floor plate region. Note that the culture of cells in the culture solution (pluripotent stem cells, cells derived from the pluripotent stem cells, etc.) is continued even during step (C).

[0139] The culture period for culturing the cells (pluripotent stem cells, cells derived from the pluripotent stem cells, etc.) in the culture medium is preferably 9 days or more and 21 days or less, and more preferably 9 days or more and 16 days or less, as the period between steps (A) and (C).

[0140] In one aspect of this embodiment, the culture of the cells from step (A) to step (C) may be carried out through a multi-step process using one or more BMP signaling inhibitors, one or more TGFβ signaling inhibitors, one or more SHH signaling activators, and one or more Wnt signaling activators as differentiation inducers. At each stage of the cell culture, the cells may be cultured in a medium containing at least one substance selected from the group consisting of a BMP signaling inhibitor, a TGFβ signaling inhibitor, an SHH signaling activator, and a Wnt signaling activator, and each stage may be adhesion culture or suspension culture. It is preferable that step (B) be carried out between the multiple steps. In one aspect of this embodiment, it is preferable that the differentiation inducer used in the culture of the cells from step (A) to step (C) further includes FGF8. That is, the culture of the cells during steps (A) to (C) may be carried out through a multi-step process using, as differentiation inducers, one or more BMP signaling inhibitors, one or more TGFβ signaling inhibitors, one or more SHH signaling activators, one or more Wnt signaling activators, and FGF8. At each step in the culture of the cells, the cells may be cultured in a medium containing at least one substance selected from a BMP signaling inhibitor, a TGFβ signaling inhibitor, an SHH signaling activator, a Wnt signaling activator, and FGF8, and each step may be adhesion culture or suspension culture. It is desirable that step (B) be carried out between the multi-step processes.

[0141] In one aspect of this embodiment, the culture of the cells during steps (A) to (C) may be carried out through a multi-step process using one or more BMP signaling inhibitors, one or more TGFβ signaling inhibitors, one or more SHH signaling activators, and one or more Wnt signaling activators as differentiation inducers. In one embodiment, one or more SHH signaling activators and one or more Wnt signaling activators may be used simultaneously with the use of one or more BMP signaling inhibitors and one or more TGFβ signaling inhibitors. Alternatively, one or more SHH signaling activators and one or more Wnt signaling activators may be used several days after the start of culture in a culture medium containing a BMP signaling inhibitor and a TGFβ signaling inhibitor. The SHH signaling activators and the Wnt signaling activators may be used in the culture medium at different times or simultaneously.

[0142] In one aspect of this embodiment, steps (A) to (C) comprise the following multi-step process for culturing pluripotent stem cells, and step (B) is desirably carried out during the culturing: (a) culturing the pluripotent stem cells on an extracellular matrix in a culture medium containing a BMP signaling inhibitor and a TGFβ signaling inhibitor; (b) culturing the cells obtained in step (a) on an extracellular matrix in a culture medium containing a BMP signaling inhibitor, a TGFβ signaling inhibitor, an SHH signaling activator, and FGF8; (c) culturing the cells obtained in step (b) on an extracellular matrix in a culture medium containing a BMP signaling inhibitor, a TGFβ signaling inhibitor, an SHH signaling activator, FGF8, and a Wnt signaling activator; and (d) culturing the cells obtained in step (c) on an extracellular matrix in a culture medium containing a BMP signaling inhibitor and a Wnt signaling activator.

[0143] In one aspect of this embodiment, step (B) is preferably performed between steps (a) to (c). In another aspect of this embodiment, the culture protocol for steps (A) to (C) may be a known protocol (e.g., Frontiers in Cell and Developmental Biology, August 2020, Volume 8, Article 729; Cell Stem Cell 28, 343-355, February 4, 2021).

[0144] In this embodiment, the extracellular matrix refers to a supramolecular structure present outside cells, and may be naturally occurring or artificial (recombinant). Examples include collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, and laminin, or fragments thereof. These extracellular matrices may be used in combination, or may be prepared from cells, such as BD Matrigel™. The extracellular matrix is ​​preferably laminin or a fragment thereof. In this embodiment, laminin is a protein with a heterotrimeric structure having one α chain, one β chain, and one γ chain. Examples include, but are not limited to, α1, α2, α3, α4, or α5 α chains, β1, β2, or β3 β chains, and γ1, γ2, or γ3 γ chains. The laminin is more preferably laminin 511, which is composed of α5, β1, and γ1 β chains. In this embodiment, the laminin may be a fragment. The laminin fragment is not particularly limited as long as it has integrin-binding activity, and may be, for example, an E8 fragment obtained by digestion with elastase. Thus, in this embodiment, an example is laminin 511E8 (preferably human laminin 511E8) described in WO2011 / 043405.

[0145] The number of days for carrying out step (a) can be 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, or more. The number of days for carrying out step (a) is preferably 1 day. Similarly, the number of days for carrying out step (b) can be 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, or more. The number of days for carrying out step (b) is preferably 2 days. Similarly, the number of days for carrying out step (c) can be 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, or more. The number of days for carrying out step (c) is preferably 4 days. Similarly, the number of days for carrying out step (d) can be 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, or more. The number of days for carrying out step (d) is preferably 5 days or more.

[0146] Pluripotent stem cells may be used after dissociation. Examples of cell dissociation methods include mechanical dissociation, dissociation using a dissociation solution having both protease and collagenase activity (e.g., Accutase™ and Accumax™), or a dissociation solution having only collagenase activity. Preferably, human pluripotent stem cells are dissociated using trypsin or a substitute thereof (e.g., TrypLE CTS (Life Technologies)). When cells are dissociated, it is desirable to add a ROCK inhibitor as appropriate after dissociation and culture the cells. When a ROCK inhibitor is added, it is sufficient to add the inhibitor and culture the cells for at least one day, more preferably one day.

[0147] <ROCK Inhibitor> In the present embodiment, the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK). Examples of the ROCK inhibitor include Y-27632 (see, for example, Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), Fasudil / HA1077 (see, for example, Uenata et al., Nature 389: 990-994 (1997)), H-1152 (see, for example, Sasaki et al., al., Pharmacol. Ther. 93:225-232 (2002)), Wf-536 (see, for example, Nakajima et al., Cancer Chemother Pharmacol. 52(4):319-324 (2003)) and derivatives thereof, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof. In addition, other low molecular weight compounds are also known as ROCK inhibitors, and such compounds or derivatives thereof can also be used in this embodiment (see, for example, U.S. Patent Application Publication Nos. 20050209261, 20050192304, 20040014755, 20040002508, 20040002507, 20030125344, 20030087919, and International Publication Nos. 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, and 2004 / 039796). In this embodiment, one or more ROCK inhibitors can be used. The ROCK inhibitor used in this embodiment may preferably be Y-27632.

[0148] The concentration of Y-27632 in the culture medium is, for example, 100 nM to 50 μM, preferably 1 μM to 10 μM, and more specifically, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, or 50 μM, but is not limited to these.

[0149] <Step (D): Differentiating Neural Cells in the Midbrain Floor Plate Region into Dopaminergic Neuronal Progenitor Cells or Their Precursor Cells> In step (D), the neural cells in the midbrain floor plate region are cultured under culture conditions that allow for differentiation into the dopaminergic neuronal progenitor cells, and differentiated into the dopaminergic neuronal progenitor cells or their precursor cells. In one aspect of this embodiment, step (D) can also be understood as a step of changing the culture conditions that allow for differentiation into neural cells in the midbrain floor plate region to culture conditions that allow for differentiation into the dopaminergic neuronal progenitor cells, and culturing the neural cells in the midbrain floor plate region to differentiate into the dopaminergic neuronal progenitor cells or their precursor cells.

[0150] The culture conditions (medium, temperature, carbon dioxide concentration, etc.) that allow differentiation into dopaminergic neural progenitor cells are described below.

[0151] The medium for culturing neural cells in the midbrain floor plate region can be prepared using a medium used for culturing animal cells as the basal medium, such as Glasgow's Minimum Essential Medium (GMEM), IMDM, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640, Fischer's medium, Neurobasal Medium (Life Technologies), and mixtures thereof. Neurobasal Medium is preferred. The medium may contain serum or may be serum-free. If necessary, the medium may contain one or more serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3′-thiolglycerol, etc., and may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, nucleic acids (e.g., dibutylyl cyclic AMP (dbcAMP)), etc. A preferred culture medium is Neurobasal Medium containing B27 supplement, ascorbic acid, and dbcAMP. Neurotrophic factors can be added to this culture medium as appropriate for culture.

[0152] <Neurotrophic Factor> In this embodiment, a neurotrophic factor is a ligand for a membrane receptor that plays an important role in the survival and maintenance of function of neurons, and examples thereof include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 / 5 (NT-4 / 5), neurotrophin-6 (NT-6), basic FGF, acidic FGF, FGF-5, epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin, and insulin-like growth factor. 1 (IGF1), Insulin-Like Growth Factor 2 (IGF2), Glia cell line-derived Neurotrophic Factor (GDNF), TGF-b2, TGF-b3, Interleukin 6 (IL-6), Ciliary Neurotrophic Factor (CNTF), and LIF.

[0153] Preferred examples of the neurotrophic factor include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 / 5 (NT-4 / 5), neurotrophin-6 (NT-6), glia cell line-derived neurotrophic factor (GDNF), TGF-b3, and ciliary neurotrophic factor (CNTF). The neurotrophic factor is preferably selected from the group consisting of GDNF, BDNF, and NT-3. It is preferable to use GDNF or BDNF, preferably GDNF and BDNF, as the neurotrophic factor. Neurotrophic factors are commercially available from, for example, Wako Co., Ltd. or R&D Systems, Inc. and can be easily used, but they may also be obtained by forced expression in cells using methods known to those skilled in the art.

[0154] The concentration of GDNF in the culture medium may be, for example, but not limited to, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, or 500 ng / mL. Preferably, it is 10 ng / mL. In one aspect of this embodiment, the concentration of GDNF in the culture medium may be 0.1 ng / mL or more and 500 ng / mL or less, or 1 ng / mL or more and 100 ng / mL or less.

[0155] The concentration of BDNF in the culture medium may be, but is not limited to, for example, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, or 500 ng / mL. Preferably, it is 20 ng / mL. The concentration of BDNF in the culture medium may be 0.1 ng / mL or more and 500 ng / mL or less, or 1 ng / mL or more and 100 ng / mL or less.

[0156] The temperature at which neural cells in the midbrain floor plate region are cultured is preferably 30°C or higher and 40°C or lower, and more preferably 35°C or higher and 38°C or lower.

[0157] The carbon dioxide concentration when culturing neural cells in the midbrain floor plate region is preferably 2% or more and 5% or less, and more preferably 4% or more and 5% or less.

[0158] The cell density when culturing neural cells in the midbrain floor plate region is preferably 50 cells / ml to 100,000 cells / ml, more preferably 500 cells / ml to 50,000 cells / ml.

[0159] The culture period for culturing the neural cells of the midbrain floor plate region in step (D) is preferably 7 days or more and 30 days or less, and more preferably 7 days or more and 24 days or less.

[0160] Step (D) may be performed by suspension culture. Suspension culture refers to culturing cells in a non-adherent state to a culture vessel, and is not particularly limited thereto. Suspension culture can be performed using a culture vessel that has not been artificially treated (e.g., coated with an extracellular matrix or the like) for the purpose of improving adhesion to the cells, or a culture vessel that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA), a nonionic surface-active polyol (Pluronic F-127, etc.), or a phospholipid-like structure (e.g., a water-soluble polymer (Lipidure) having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit).

[0161] In one aspect of this embodiment, step (D) is preferably carried out by selecting and recovering midbrain floor plate cells from the neural cells in the midbrain floor plate region, and culturing the recovered midbrain floor plate cells under culture conditions that allow them to be induced to differentiate into the dopaminergic neural progenitor cells.

[0162] The method for selecting and recovering mesencephalic floor plate cells from the neural cells in the mesencephalic floor plate region can be carried out by using a substance that specifically binds to a marker molecule present on the cell surface of the mesencephalic floor plate cells to select them from a cell population. The substance that binds to the marker molecule can be an antibody or an aptamer, preferably an antibody or an antigen-binding fragment thereof.

[0163] In this embodiment, the antibody may be a polyclonal or monoclonal antibody, and these antibodies can be produced using techniques well known to those skilled in the art (Current protocols in Molecular Biology, edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.12-11.13). On the other hand, monoclonal antibodies can be obtained from hybridoma cells prepared by cell fusion between spleen cells obtained from a non-human animal immunized with a marker molecule and myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11). Examples of antigen-binding fragments of antibodies include portions of antibodies (e.g., Fab fragments) and synthetic antibody fragments (e.g., single-chain Fv fragments "ScFv"). Fab and F(ab) ２ Fragments of antibodies, such as fragments, can also be produced by well-known methods of genetic engineering.

[0164] For the purpose of recognizing or isolating midbrain floor plate cells expressing a marker molecule, the binding substance may be bound or conjugated to a detectable substance such as a fluorescent label, a radioactive label, a chemiluminescent label, an enzyme, biotin, or streptavidin, or to a substance that enables isolation and extraction, such as protein A, protein G, beads, or magnetic beads. The binding substance may also be indirectly labeled. This can be done using various methods known to those skilled in the art, such as a method using a pre-labeled antibody (secondary antibody) that specifically binds to the antibody.

[0165] Methods for detecting target cells, such as midbrain floor plate cells, include the use of a flow cytometer or a protein chip.

[0166] Examples of methods for extracting target cells, such as midbrain floor plate cells, include a method in which particles are attached to a binding substance and then precipitated, a method in which magnetic beads are used to magnetically select cells (e.g., MACS), a method in which a cell sorter is used with fluorescent labels, or a method in which a carrier on which an antibody or the like is immobilized (e.g., a cell concentration column) is used.

[0167] In this embodiment, examples of marker molecules for midbrain floor plate cells that can be used to select and recover target cells include CORIN, LRTM1, CD142, etc. Midbrain floor plate cells are a concept encompassed by nervous system cells in the midbrain floor plate region.

[0168] In one aspect of this embodiment, it is preferable to further include, between steps (C) and (D), a step (E) of recovering the neural cells of the midbrain floor plate region. Here, "recovery" refers to a step of collecting the cells obtained in step (C). For example, in the case of adherent culture or when cell aggregates are formed in suspension culture, recovery also includes procedures such as mechanical dispersion (e.g., pipetting or scraping with a scraper) or cell dispersion treatment (e.g., treatment with enzymes such as trypsin, collagenase, or papain, or chelating agents such as ethylenediaminetetraacetic acid) to detach the cells from the culture vessel or disperse the aggregated cells. Using the cells recovered in step (E), the desired midbrain floor plate cells can be selected from the population of neural cells in the midbrain floor plate region, and the cell population can be purified, as described above.

[0169] In one embodiment of the selection of midbrain floor plate cells in step (D), a substance that specifically binds to Corin can be used to select neural cells in the midbrain floor plate region from the cell population. Specific means for this can be the same as the method for recovering midbrain floor plate cells described above.

[0170] The dopaminergic neural progenitor cells and / or their precursor cells (hereinafter, sometimes referred to as "dopamine neural progenitor cells, etc.") produced by the method for producing dopaminergic neural progenitor cells or their precursor cells according to this embodiment can be used as a transplant tissue. Examples of the transplant tissue include a transplant tissue for transplantation into the brain of a Parkinson's disease patient. The number of cells, such as the dopaminergic neural progenitor cells, in the transplant tissue is not particularly limited, but may be, for example, 5 x 10 ５ 3x10 pieces or more ７ Examples of embodiments include transplanting cells in an amount of 100 or less. The shape of the tissue for transplantation may be a sheet, a sphere, or a shape that matches the site to which it is to be transplanted. The tissue for transplantation may also be in the form of a suspension.

[0171] From the viewpoint of preventing rejection reactions, it is preferable that the HLA genotype of the cells contained in the transplant tissue is identical or substantially identical to the HLA genotype of the recipient individual.

[0172] Transplantation of the transplant tissue containing the dopamine neural progenitor cells or the like into the diseased site can be carried out by, for example, Nature Communication, 11, 3369 (2020), Nature Neuroscience, 2, 1137 (1999), or N Engl J Med.; 344: 710-9 (2001).

[0173] Examples of the present invention are described below, but the present invention is not limited to these. <Experiment 1> <Undifferentiated State Maintenance Culture> Prior to the differentiation-inducing culture described below, iPS cells were cultured while maintaining their undifferentiated state. Specifically, two types of culture conditions were set for the final stage of the undifferentiated state maintenance process under the conditions of culture experiments A, B, and C, and a comparison study was conducted.

[0174] <Culture Experiment A> A research iPS cell line (official name: 1231A3) was cultured in a medium with the following composition. SAG (N-Methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane, final concentration 300 nM) was added to the medium (pretreatment) for one day from one day before the start of differentiation culture (Day -1) to the day of differentiation initiation (Day 0), and a cell group with high induction efficiency (cell group A1) was established. Additionally, for comparison, an untreated cell group (cell group A2) was established, cultured under the same conditions as cell group A1, except that SAG was not added to the medium. Regarding the medium in culture experiment A, AK03N basal medium manufactured by Ajinomoto Co., Inc. was used before the start of differentiation, and GMEM containing 8% KSR was used as the basal medium from the start of differentiation (Day 0) onwards.

[0175] <Culture Experiment B> An iPS cell line (QHJI01s04 line) for clinical research was used and cultured under similar culture conditions to cell group A2 in culture experiment A. In culture experiment B, cell groups B1 and B2 were established, each with a different cell culture period. Cell group B1 was a cell group with low differentiation induction efficiency, while cell group B2 was a cell group with high differentiation induction efficiency.

[0176] <Culture Experiment C> A research iPS cell line (official name: 201B7) was used and cultured under culture conditions similar to those of cell group A2 in culture experiment A. In culture experiment C, cell groups C1 and C2 were established using different medium components. Cell group C1 was a cell group with low differentiation induction efficiency, while cell group C2 was a cell group with high differentiation induction efficiency.

[0177] <<Culture for Induction of Differentiation into Neural Cells of the Midbrain Floor Plate Region>> According to a prescribed protocol, differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region was induced for each of the six cell groups prepared in the above culture experiments A, B, and C. That is, on day 0 of differentiation initiation, A-83-01 (a TGFβ signaling inhibitor, a type of SMAD signaling inhibitor, final concentration 0.5 μM) and LDN-193189 (a BMP signaling inhibitor, a type of SMAD signaling inhibitor, final concentration 0.1 μM) were added to the medium. One day after differentiation initiation, Purmorphamine (an SHH signaling activator, final concentration 2 μM) and FGF8 (final concentration 100 ng / mL) were added to the medium. Three days after differentiation initiation, CHIR99021 (a Wnt signaling activator, final concentration 3 μM) was added. The addition of A-83-01, Purmorphamine, and FGF8 was terminated 7 days after the start of differentiation. From the start of differentiation to the 13th day, the entire medium was replaced every 24 hours, and when the medium composition was changed according to the above protocol, the culture was switched to a medium with a different composition at the time of medium replacement. On the 12th day after the start of differentiation, the proportion of Corin-positive cells in each cell group was measured using a cell sorter (BD FACSJazz, BD Accuri, or Furukawa PERFLOW Sort). The manufacturers of each reagent are as follows: A-83-01: Manufactured by Wako LDN-193189: Manufactured by STEMGENT Purmorphamine: Manufactured by Wako FGF8: Manufactured by Wako CHIR99021: Manufactured by Wako

[0178] <NT-3 Concentration Analysis> Immediately after the start of differentiation-inducing culture (i.e., immediately after the start of differentiation), a portion of the culture supernatant from each cell group was collected every 24 hours or 48 hours, and the NT-3 concentration was analyzed. The timing of collection of the culture supernatant was set at a predetermined time within a 24-hour period (1 day), coinciding with the timing of the medium change described above. When collecting the culture supernatant, the entire medium was temporarily collected in a container separate from the incubator, and a portion of it was collected while the entire medium was thoroughly stirred. The results are shown in Figures 1 to 3. Bioplex (registered trademark) manufactured by BioRad was used for the NT-3 concentration analysis.

[0179] <<Results>> <Culture Experiment A>> In cell group A1, the percentage of Corin-positive cells on day 12 of differentiation-inducing culture was 90%. On the other hand, in cell group A2, the percentage of Corin-positive cells on day 12 of differentiation-inducing culture was 41%. Regarding the analysis of NT-3 concentration in the culture supernatant, in cell group A1, NT-3 was not detected in the culture supernatant samples collected 1 and 5 days after the start of differentiation, but was specifically increased in the sample collected 3 days later (Figure 1). On the other hand, in cell group A2, NT-3 was not detected in the culture supernatant samples collected on either day.

[0180] <Culture Experiment B> In cell group B1, the percentage of Corin-positive cells on day 12 of differentiation-inducing culture was 29%. On the other hand, in cell group B2, the percentage of Corin-positive cells on day 12 of differentiation-inducing culture was 65%. Regarding the analysis of NT-3 concentration in the culture supernatant, in cell group B2, NT-3 was not detected in the culture supernatant samples collected 1, 4, and 5 days after the start of differentiation, but was specifically increased in the culture supernatant samples collected 2 and 3 days later (Figure 2). On the other hand, in cell group B1, NT-3 was not detected in the culture supernatant samples collected on any day.

[0181] <Culture Experiment C> In cell group C1, the percentage of Corin-positive cells on day 12 of differentiation-inducing culture was 24%. On the other hand, in cell group C2, the percentage of Corin-positive cells on day 12 of differentiation-inducing culture was 83%. Regarding the concentration of NT-3 in cell group C2, it was not detected in the culture supernatant samples collected 1 and 5 days after the start of differentiation, but it specifically increased in the samples collected on days 2, 3, and 4 (Figure 3). On the other hand, in cell group C1, NT-3 was not detected in the culture supernatant samples collected on any day.

[0182] Based on the results of the above-mentioned culture experiments A, B, and C, it is considered preferable to set the reference concentration in the first method to, for example, 10 pg / ml. Furthermore, it is considered preferable to set the reference rate of change in the second method to, for example, 5 pg / ml-day. It is desirable to set the reference concentration depending on the production method, the type and strain of cells used, and the production process to be carried out.

[0183] <Culture Experiment D> Culture was performed under the same undifferentiation maintenance culture conditions and differentiation induction conditions as in Culture Experiment C. Cell group D1 was cultured under the same conditions (deviation conditions) as cell group C1, and cell group D2 was cultured under the same conditions (standard conditions) as cell group C2. In Culture Experiment D, single-cell gene expression analysis was performed on cell groups D1 and D2 to analyze NT-3 gene expression. Cells were collected at the start of differentiation (Day 0) and on days 4, 8, and 12 after the start of differentiation, and a single-cell gene expression library was prepared. Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics) was used to prepare the single-cell gene expression library. A Novaseq 6000 was used for sequencing analysis.

[0184] <Results> The results of the single-cell gene expression analysis are shown in Figure 4. In cell group D2, the percentage of NT-3 gene (NTF3)-positive cells on day 4 of differentiation-inducing culture was 76%, while in cell group D1, the percentage of NTF3-positive cells on day 4 of differentiation-inducing culture was 60%. Furthermore, the counts per million (CPM), an indicator of average RNA expression level, was 49 in cell group D2, while it was 28 in cell group D1. As described above, even at the mRNA level, NT-3 expression correlated with the NT-3 content in the culture supernatant. Furthermore, it was found that NT-3 expression was not observed only in a subpopulation, but was temporarily observed in the majority of cells during the differentiation process into target cells following differentiation induction. This demonstrates that NT-3 is suitable as a monitoring molecule for early assessment of differentiation induction.

[0185] From the above results, it was found that by monitoring the concentration of NT-3 secreted into the culture supernatant from pluripotent stem cells, i.e., secreted into the culture supernatant during the differentiation process from pluripotent stem cells to neural cells of the midbrain floor plate region, it is possible to determine the differentiation potential of cells in the culture medium during the differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region at an early stage of differentiation induction.

[0186] <Experiment 2> <Culture for maintaining undifferentiated state> Prior to differentiation-inducing culture, iPS cells were cultured while maintaining their undifferentiated state. Specifically, one or more culture conditions were set for the final stage of the undifferentiated state maintenance step under the conditions of culture experiments E and F, and a comparative study was conducted.

[0187] <Culture Experiment E> An iPS cell line established from adult peripheral blood mononuclear cells using a Sendai virus vector (CytoTune™-2.0; ID Pharma) was cultured under differentiation induction conditions similar to those for cell group A2 in culture experiment A.

[0188] <Culture Experiment F> An iPS cell line (official name: 201B7) was used and cultured under culture conditions similar to those of cell group A2 in culture experiment A. In culture experiment F, cell groups F1 and F2 were established, each containing different medium components.

[0189] <<Culture for inducing differentiation into neural cells in the midbrain floor plate region>> Following the same protocol as in Experiment 1 above, the three types of cell groups prepared in culture experiments E and F above were each induced to differentiate from pluripotent stem cells into neural cells in the midbrain floor plate region.

[0190] <NT-3 Concentration Analysis> Immediately after the start of differentiation-inducing culture (i.e., immediately after the start of differentiation), a portion of the culture supernatant from each cell group was collected every 24 hours or 48 hours and analyzed for NT-3 concentration. At this time, the culture supernatant obtained in the above-mentioned culture experiment D was also analyzed for NT-3 concentration. The results are shown in Figures 5 to 7. Bioplex (registered trademark) manufactured by BioRad was used for the analysis of NT-3 concentration in culture experiment E. Digital ELISA was used for the analysis of NT-3 concentration in culture experiments D and F.

[0191] <Results> <Culture Experiment E> In the cell group used in Culture Experiment E, the percentage of Corin-positive cells on day 12 of differentiation-inducing culture was 44%. NT-3 concentration was not detected in the culture supernatant samples collected 1 to 4 days and 9 to 12 days after the start of differentiation, but was specifically increased in the samples collected 5 to 8 days later (Figure 5).

[0192] <Culture Experiments D and F> In cell group D1, the percentage of Corin-positive cells on day 12 of differentiation-inducing culture was 34%. On the other hand, in cell group D2, the percentage of Corin-positive cells on day 12 of differentiation-inducing culture was 58%. Regarding the concentration of NT-3, it was not detected in the culture supernatant samples collected one day after the start of differentiation in cell groups D1 and D2, but gradually increased from day 2 onwards (Figure 6). Furthermore, cell group D2 showed a tendency for the concentration of NT-3 to be higher than cell group D1, which correlated with the percentage of Corin-positive cells.

[0193] In cell group F1, the percentage of Corin-positive cells on day 12 of differentiation-inducing culture was 57%. On the other hand, in cell group D2, the percentage of Corin-positive cells on day 12 of differentiation-inducing culture was 67%. The concentration of NT-3 gradually increased in cell groups F1 and F2 after day 1 of differentiation (Figure 7). Furthermore, cell group F2 tended to have a higher concentration of NT-3 than cell group F1, which correlated with the percentage of Corin-positive cells.

[0194] The results of Experiment 2 also showed that by monitoring the concentration of NT-3 secreted into the culture supernatant from pluripotent stem cells, it is possible to determine the differentiation potential of cells in the culture medium during differentiation from pluripotent stem cells into neural cells in the midbrain floor plate region at an early stage of differentiation induction.

[0195] <<Culture for inducing differentiation into dopaminergic neural progenitor cells>> Using each of the six cell groups in the above-described culture experiments A, B, and C, culture for inducing differentiation into dopaminergic neural progenitor cells was performed by the following method: On day 12 after the start of differentiation induction, flow cytometry analysis was performed using an anti-Corin antibody to recover Corin-positive cells. The collected Corin-positive cells were transferred to Prime Surface 96U bottom plates (Sumitomo Bakelite) at 20,000 cells / well and cultured in suspension using basal medium B (Neurobasal medium (Invitrogen) supplemented with B27 Supplement without vitamin A (Invitrogen), 20 ng / mL BDNF, 10 ng / mL GDNF, 200 mM Ascorbic acid, and 0.4 mM dbcAMP (Sigma)). The initial medium used was medium supplemented with 30 μM Y-27632, and when half of the medium was replaced every three days, medium without Y-27632 was used. The Corin-positive cells are cultured in suspension for 16 days (day 28) after collection, and then stained for FoxA2 and Nurr1 (both midbrain markers) to confirm whether they have differentiated into dopaminergic neural progenitor cells.

[0196] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0197] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include any modifications within the scope of the claims and meanings equivalent to the claims.

Claims

1. A method for determining the differentiation potential of cells in a culture medium in differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region, comprising: measuring the concentration of NT-3 in a culture supernatant of a culture solution obtained by culturing the pluripotent stem cells in a medium containing an inducer of differentiation into neural cells of the midbrain floor plate region; comparing the measured concentration of NT-3 with a reference concentration; determining that the cells in the culture medium are capable of differentiating into neural cells of the midbrain floor plate region when the concentration of the NT-3 is equal to or greater than the reference concentration; Including, A method for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region, wherein the culture supernatant is a culture supernatant collected from the culture medium any time between 48 hours and 240 hours after the start of culturing the pluripotent stem cells.

2. A method for determining the differentiation potential of cells in a culture medium in differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region, comprising: measuring the concentrations of NT-3 in each of a first culture supernatant and a second culture supernatant in a culture solution obtained by culturing the pluripotent stem cells in a medium containing an inducer of differentiation into neural cells of the midbrain floor plate region; determining a rate of change in the concentration of NT-3 from the concentration of NT-3 in the first culture supernatant and the concentration of NT-3 in the second culture supernatant; comparing the absolute value of the rate of change in the concentration of NT-3 with a reference rate of change; determining that the cells in the culture medium are capable of differentiating into neural cells of the midbrain floor plate region when the absolute value of the rate of change in the concentration of NT-3 is equal to or greater than the reference rate of change; Including, the first culture supernatant is a culture supernatant collected from the culture medium at any time between 48 hours and 192 hours after the start of culturing the pluripotent stem cells; A method for determining the differentiation potential of cells in a culture medium in the differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region, wherein the second culture supernatant is a culture supernatant collected from the culture medium any time between 24 hours and 96 hours before collection of the first culture supernatant or any time between 24 hours and 96 hours after collection of the first culture supernatant.

3. A method for determining the differentiation potential of cells in a culture medium in differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region, comprising: measuring the concentrations of NT-3 in each of a first culture supernatant, a second culture supernatant, and a third culture supernatant in a culture solution obtained by culturing the pluripotent stem cells in a medium containing an inducer of differentiation into neural cells of the midbrain floor plate region; comparing the concentration of NT-3 in the first culture supernatant, the concentration of NT-3 in the second culture supernatant, and the concentration of NT-3 in the third culture supernatant; determining that the cells in the culture medium are capable of differentiating into neural cells of the midbrain floor plate region when the concentration of NT-3 in the first culture supernatant is higher than the concentrations of NT-3 in the second culture supernatant and the third culture supernatant; Including, the first culture supernatant is a culture supernatant collected from the culture medium at any time between 48 hours and 192 hours after the start of culturing the pluripotent stem cells; the second culture supernatant is collected from the culture medium at any time between 24 hours and 96 hours before collection of the first culture supernatant; A method for determining the differentiation potential of cells in a culture medium in the differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region, wherein the third culture supernatant is a culture supernatant collected from the culture medium any time between 24 hours and 96 hours after collection of the first culture supernatant.

4. A method for determining the differentiation potential of cells in a culture medium in the differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region described in any one of claims 1 to 3, wherein the differentiation inducer into neural cells of the midbrain floor plate region contains at least one SMAD signaling inhibitor.

5. A method for determining the differentiation potential of cells in a culture medium in the differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region, as described in claim 4, wherein the SMAD signaling inhibitor comprises at least one BMP signaling inhibitor and at least one TGFβ signaling inhibitor.

6. The BMP signaling inhibitor comprises at least one selected from the group consisting of LDN-193189, Noggin, DMH1, Chordin, Follistatin, K02288, LDN-214117, LDN-212854, ML347, and Dorsomorphin; The TGFβ signaling inhibitors include SB-431542, A-83-01, Lefty-1, Lefty-2, Galunisertib, SB-202190, SB-525334, SB-505124, NPC30345, Prifenidone, GW788388, E-616452, SD208, TP0427736, and BIB The method for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region according to claim 5, wherein the culture medium contains at least one selected from the group consisting of F-0775, LY3200882, Vactosertib, ITD-1, SD093, SD908, LY2109761, LY364947, and LY580276.

7. A method for determining the differentiation potential of cells in a culture medium in the differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region described in any one of claims 1 to 3, wherein the differentiation inducer into neural cells of the midbrain floor plate region comprises an SHH signaling active substance and / or a Wnt signaling active substance.

8. A method for determining the differentiation potential of cells in a culture medium in the differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region, as described in claim 7, wherein the differentiation inducer into neural cells of the midbrain floor plate region comprises an SHH signaling active substance and a Wnt signaling active substance.

9. The method for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region according to claim 7, wherein the SHH signaling active substance comprises at least one member selected from the group consisting of SHH and fragments and modified forms thereof, SHH receptor, SHH receptor agonist, Hh-Ag1.5, smoothened agonist, 20a-hydroxycholesterol, purmorphamine, and SAG.

10. The method for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region according to claim 7, wherein the Wnt signaling active substance comprises at least one substance selected from the group consisting of WNT3A and a GSK-3β inhibitor.

11. The method for determining the differentiation potential of cells in a culture medium during differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region according to claim 10, wherein the GSK-3β inhibitor comprises at least one selected from the group consisting of CHIR99021, BIO, CHIR98014, SKL2001, SB216763, GSK-3β inhibitor VII, and L803-mts.

12. A method for determining the differentiation potential of cells in a culture medium in the differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region described in any one of claims 1 to 3, wherein the pluripotent stem cells are iPS cells or ES cells.

13. A method for producing dopaminergic neural progenitor cells or their precursor cells from pluripotent stem cells, comprising: (A) starting the culture of the pluripotent stem cells under culture conditions that allow differentiation into neural cells in the midbrain floor plate region; (B) performing a method for determining the differentiation potential of cells in a culture solution in the differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region according to any one of claims 1 to 3, between 0 and 288 hours after the start of culturing the pluripotent stem cells; (C) determining to continue culturing the cells in the culture solution determined in step (B) to be capable of differentiating into neural cells of the midbrain floor plate region, thereby differentiating the cells in the culture solution into neural cells of the midbrain floor plate region; (D) culturing the neural cells in the midbrain floor plate region under culture conditions that allow differentiation into the dopaminergic neural progenitor cells, thereby differentiating the cells into the dopaminergic neural progenitor cells or their precursor cells; A method for producing dopaminergic neural progenitor cells or precursor cells thereof from pluripotent stem cells, comprising:

14. Between step (C) and step (D), (E) recovering the neural cells of the midbrain floor plate region obtained in the step (C); A method for producing dopaminergic neural progenitor cells or progenitor cells thereof from the pluripotent stem cells according to claim 13, further comprising:

15. 14. A method for producing dopaminergic neural progenitor cells or their precursor cells from pluripotent stem cells according to claim 13, wherein step (D) is carried out by selecting and recovering midbrain floor plate cells from the neural cells of the midbrain floor plate region, and culturing the recovered midbrain floor plate cells under culture conditions that allow them to be induced to differentiate into the dopaminergic neural progenitor cells.