Method for non-invasively monitoring state of cells within culture broth in differentiation of pluripotent stem cells into neural cells of midbrain floor plate region

Abstract

Provided is a method for non-invasively monitoring the state 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 non-invasively monitoring the state 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 a cell-secreted factor in the culture supernatant of 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 cell-secreted factor concentration with a standard concentration, and a step for assessing that the cells within the culture broth can differentiate into neural cells of the midbrain floor plate region if the cell-secreted factor concentration is equal to or greater than the standard concentration; and the culture supernatant is collected from the culture broth at any time point within 336 hours after the start of culture of the pluripotent stem cells.

Description

A method for non-invasively monitoring the state of cells in culture during differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region

[0001] The present invention relates to a method for non-invasively monitoring the state of cells in a culture medium during 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, 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] Details regarding detectable substances present in the culture supernatant during the early stages of producing dopaminergic neural progenitor cells from pluripotent stem cells via neural progenitor cells and neural cells in the midbrain floor plate region are unknown. Furthermore, for example, neurotrophin-3 (NT-3), a member of the nerve growth factor (NGF) family, 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, it is not 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 neural cells in the midbrain floor plate region, nor is it known whether NT-3 is secreted into the culture supernatant during the differentiation process of neural cells in the midbrain floor plate region.

[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 steps and the long time required to obtain the target cells. Therefore, a method for producing dopaminergic neural progenitor cells is needed to predict, at an early stage of differentiation induction, whether the differentiation induction is progressing smoothly and whether a normal lot containing a certain percentage of target cells suitable for human transplantation can be obtained. Furthermore, an indicator for noninvasively monitoring the state of cells during the differentiation induction process is needed to confirm whether the differentiation process is progressing normally over time. Here, the "state of cells" includes the ability to differentiate into neural cells in 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.

[0007] The present invention has been made in view of the above circumstances, and the problem that the present invention aims to solve is to provide a method for non-invasively monitoring the state of cells during the process of differentiation from pluripotent stem cells to dopaminergic neural progenitor cells, 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 by monitoring the concentration of a specific cell-secreted factor secreted into the culture supernatant during the process of differentiation from pluripotent stem cells into dopaminergic neural progenitor cells, it is possible to non-invasively monitor the state of cells during the process of differentiation from pluripotent stem cells into dopaminergic neural progenitor cells at an early stage of differentiation induction, and thus completed the present invention.

[0009] [1] A first aspect of the present invention is a method for non-invasively monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region, comprising the steps of: measuring the concentration of a cell-secreted factor in a 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; comparing the measured concentration of the cell-secreted factor with a reference concentration; and 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 cell-secreted factor is equal to or greater than the reference concentration, wherein the culture supernatant is collected from the culture medium at any time within 336 hours after the start of culturing the pluripotent stem cells.

[0010] [2] A second aspect of the present invention is a method for non-invasively monitoring the state 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 cell-secreted factors 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 the concentration of the cell-secreted factor from the concentrations of the cell-secreted factor in the first culture supernatant and the second culture supernatant; comparing the absolute value of the rate of change in the concentration of the cell-secreted factor 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 the concentration of the cell-secreted factor is equal to or greater than the standard rate of change; wherein the first culture supernatant is a culture supernatant collected from the culture medium at any time within 336 hours after the start of culturing the pluripotent stem cells, The second culture supernatant is a culture supernatant collected from the culture medium at any time between 24 hours and 72 hours before collection of the first culture supernatant or at any time between 24 hours and 72 hours after collection of the first culture supernatant.

[0011] [3] A third aspect of the present invention is a method for non-invasively monitoring the state 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 a cell-secreted factor in each of 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 concentration of the cell-secreted factor 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 the cell-secreted factor in the first culture supernatant is higher than the concentration of the cell-secreted factor in the second culture supernatant and the third culture supernatant. The first culture supernatant is a culture supernatant collected from the culture medium at any time within 336 hours after the start of culturing the pluripotent stem cells, the second culture supernatant is a culture supernatant collected from the culture medium at any time between 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 at any time between 24 hours and 72 hours after the collection of the first culture supernatant.

[0012] [4] In any one of [1] to [3] above, the concentration of the cell-secreted factor may be the amount of the cell-secreted factor appearing in the culture medium per unit time.

[0013] [5] In the above [1] or [4], the reference concentration may be the minimum concentration at which differentiation is successful.

[0014] [6] In any of [1] to [5] above, the cell-secreted factor may be any one or more selected from the group consisting of Gas6, SerpinF1, TFF3, WFDC2, Cripto, CD30, B2M, CXCL6, IGFBP6, HGFR, SPARC, TRANCE, PDGF-AA, FGF13, Kallikrein6, MMP2, RAC2, TKT, NPTX1, and NT-3.

[0015] [7] In any of the above [1] to [6], the pluripotent stem cells may be iPS cells or ES cells.

[0016] [8] In any of [1] to [7] above, the step of measuring the concentration of the cell-secreted factor may include measuring the concentration of one or more of the cell-secreted factors that are expected to appear within 336 hours after the start of culture multiple times.

[0017] [9] In the above-mentioned [8], the cell-secreted factor expected to appear within 336 hours after the initiation of the culture may be any one or more selected from the group consisting of Gas6, SerpinF1, TFF3, WFDC2, Cripto, CD30, B2M, CXCL6, IGFBP6, HGFR, SPARC, TRANCE, PDGF-AA, FGF13, Kallikrein6, MMP2, RAC2, TKT, NPTX1, and NT-3.

[0018]

[10] In any of [1] to [9] above, the method may further comprise the step of determining whether or not there is an abnormality in the cells in the culture medium.

[0019]

[11] A fourth aspect of the present invention is a method for producing dopaminergic neural progenitor cells or precursor cells thereof from pluripotent stem cells, comprising: (A) initiating 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 non-invasively monitoring the state of cells in a culture medium during differentiation of the pluripotent stem cells into neural cells of the midbrain floor plate region according to any one of [1] to

[10] , between 0 and 336 hours after initiation of culture of the pluripotent stem cells; (C) deciding to further continuously culture 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, thereby differentiating the cells in the culture medium into neural cells of the midbrain floor plate region; and (D) culturing the neural cells of the midbrain floor plate region under culture conditions that allow differentiation into the dopaminergic neural progenitor cells, thereby differentiating them into the dopaminergic neural progenitor cells or precursor cells thereof.

[0020]

[12] In the above

[11] , the method may further comprise, between step (C) and step (D), the step (E) of recovering the neural cells in the midbrain floor plate region.

[0021]

[13] In the above

[11] or

[12] , step (D) may be carried out by selecting and recovering mesencephalic floor plate cells differentiated from neural cells in the mesencephalic floor plate region, and culturing the recovered mesencephalic floor plate cells under culture conditions that allow them to be induced to differentiate into the dopaminergic neural progenitor cells.

[0022] According to the present invention, it is possible to provide a method for non-invasively monitoring the state of cells during the process of differentiation from pluripotent stem cells to dopaminergic neural progenitor cells, which enables determination at an early stage of differentiation induction from pluripotent stem cells to dopaminergic neural progenitor cells. Furthermore, since the present invention enables non-invasive monitoring of the state of cells at an early stage of differentiation induction, it is possible, for example, to discontinue the culture of a culture lot with poor differentiation potential, thereby reducing production costs.

[0023] FIG. 1A is a graph showing the change in Gas6 concentration in the culture supernatant over time in cell group x1 with high differentiation efficiency and cell group x2 with low differentiation efficiency. The horizontal axis represents the number of days of culture for differentiation induction, and the vertical axis represents the Gas6 concentration in the supernatant. The data show that the Gas6 concentration increases under conditions of high differentiation induction efficiency. FIG. 1B is a graph showing the change in Gas6 concentration in the culture supernatant over time in cell group w1 with high differentiation efficiency and cell group w2 with low differentiation efficiency. The horizontal axis represents the number of days of culture for differentiation induction, and the vertical axis represents the Gas6 concentration in the supernatant. The data show that the Gas6 concentration increases under conditions of high differentiation induction efficiency. FIG. 1C is a graph showing the correlation between differentiation efficiency (%) and the Gas6 concentration in the supernatant one day after the start of differentiation induction in multiple differentiation culture experiments. The horizontal axis represents differentiation efficiency (%), and the vertical axis represents the Gas6 concentration in the supernatant one day after the start of differentiation induction. The data suggest that there is a correlation between differentiation induction efficiency and Gas6 concentration. 2A is a graph showing the change in SerpinF1 concentration in the culture supernatant over time for cell group x1 with high differentiation efficiency and cell group x2 with low differentiation efficiency. The horizontal axis represents the number of days of culture for differentiation induction, and the vertical axis represents the SerpinF1 concentration in the supernatant. The data show that the SerpinF1 concentration increases under conditions of high differentiation induction efficiency. FIG. 2B is a graph showing the change in SerpinF1 concentration in the culture supernatant over time for cell group w1 with high differentiation efficiency and cell group w2 with low differentiation efficiency. The horizontal axis represents the number of days of culture for differentiation induction, and the vertical axis represents the SerpinF1 concentration in the supernatant. The data show that the SerpinF1 concentration increases under conditions of high differentiation induction efficiency. FIG. 2C is a graph showing the correlation between differentiation efficiency (%) and the SerpinF1 concentration in the supernatant 8 days after the start of differentiation induction in multiple differentiation culture experiments. The horizontal axis represents differentiation efficiency (%), and the vertical axis represents the concentration of SerpinF1 in the supernatant 8 days after the start of differentiation induction. This data suggests a correlation between differentiation induction efficiency and SerpinF1 concentration. Figure 3A is a graph showing the change in TFF3 concentration in the culture supernatant over time for cell group x1 with high differentiation efficiency and cell group x2 with low differentiation efficiency. The horizontal axis represents the number of days of culture for differentiation induction, and the vertical axis represents the TFF3 concentration in the supernatant. This data shows that the TFF3 concentration increases under conditions of high differentiation induction efficiency. Figure 3B is a graph showing the change in TFF3 concentration in the culture supernatant over time for cell group w1 with high differentiation efficiency and cell group w2 with low differentiation efficiency.The horizontal axis represents the number of days of culture for differentiation induction, and the vertical axis represents the TFF3 concentration in the supernatant. This data shows that the TFF3 concentration increases under conditions with high differentiation induction efficiency. Figure 3C is a graph showing the correlation between differentiation efficiency (%) and the TFF3 concentration in the supernatant 11 days after the start of differentiation induction in multiple differentiation culture experiments. The horizontal axis represents differentiation efficiency (%), and the vertical axis represents the TFF3 concentration in the supernatant 11 days after the start of differentiation induction. This data suggests that there is a correlation between differentiation induction efficiency and TFF3 concentration. Figure 4 is a graph showing the change in Cripto concentration in the culture supernatant over time in cell group x1 with high differentiation efficiency and cell group x2 with low differentiation efficiency. The horizontal axis represents the number of days of culture for differentiation induction, and the vertical axis represents the Cripto concentration in the supernatant. This data shows that the Cripto concentration increases under conditions with high differentiation induction efficiency. Figure 5A is a graph showing the change in WFDC2 concentration in the culture supernatant over time for cell group x1 with high differentiation efficiency and cell group x2 with low differentiation efficiency. The horizontal axis represents the number of days of differentiation induction culture, and the vertical axis represents the WFDC2 concentration in the supernatant. The data show that the WFDC2 concentration increases under conditions of high differentiation induction efficiency. Figure 5B is a graph showing the correlation between the WFDC2 concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 in cell groups with different differentiation efficiencies in multiple differentiation culture experiments. The horizontal axis represents differentiation induction efficiency (%), and the vertical axis represents WFDC2 concentration per unit cell number. The data show that the differentiation induction efficiency and WFDC2 concentration per unit cell number are correlated. Figure 6A is a graph showing the change in CD30 concentration in the culture supernatant over time for cell group x1 with high differentiation efficiency and cell group x2 with low differentiation efficiency. 6B is a graph showing the correlation between the CD30 concentration in the culture supernatant on Day 0 of differentiation induction and the differentiation induction efficiency on Day 13 of differentiation induction in cell groups with different differentiation efficiencies in multiple differentiation culture experiments. The horizontal axis represents the differentiation induction efficiency (%), and the vertical axis represents the CD30 concentration per unit cell number. The data shows that the differentiation induction efficiency and the CD30 concentration per unit cell number are correlated.7A is a graph showing the change in B2M concentration in the culture supernatant over time in a cell group x1 with high differentiation efficiency and a cell group x2 with low differentiation efficiency. The horizontal axis represents the number of days of differentiation induction culture, and the vertical axis represents the B2M concentration in the supernatant. The data show that the B2M concentration increases under conditions of high differentiation induction efficiency. FIG. 7B is a graph showing the correlation between the B2M concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 in cell groups with different differentiation efficiencies in multiple differentiation culture experiments. The horizontal axis represents differentiation induction efficiency (%), and the vertical axis represents the B2M concentration per unit cell number. The data show that the differentiation induction efficiency and the B2M concentration per unit cell number are correlated. FIG. 8 is a graph showing the correlation between the CXCL6 concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 in cell groups with different differentiation efficiencies in multiple differentiation culture experiments. The horizontal axis represents the differentiation induction efficiency (%), and the vertical axis represents the CXCL6 concentration per unit cell number. This data shows a correlation between the differentiation induction efficiency and the CXCL6 concentration per unit cell number. Figure 9 is a graph showing the correlation between the IGFBP6 concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 in cell groups with different differentiation efficiencies in multiple differentiation culture experiments. The horizontal axis represents the differentiation induction efficiency (%), and the vertical axis represents the IGFBP6 concentration per unit cell number. This data shows a correlation between the differentiation induction efficiency and the IGFBP6 concentration per unit cell number. Figure 10 is a graph showing the correlation between the HGFR concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 in cell groups with different differentiation efficiencies in multiple differentiation culture experiments. 11 is a graph showing the correlation between the SPARC concentration in the culture supernatant on Day 0 of differentiation induction and the differentiation induction efficiency on Day 13 of differentiation induction in cell groups with different differentiation efficiencies in multiple differentiation culture experiments. The horizontal axis is the differentiation induction efficiency (%), and the vertical axis is the SPARC concentration per unit number of cells, and the data shows that the correlation between the differentiation induction efficiency and the SPARC concentration per unit number of cells.12 is a graph showing the correlation between the TRANCE concentration in the culture supernatant on differentiation induction Day 0 and the differentiation induction efficiency on differentiation induction Day 13 in cell groups with different differentiation efficiencies in multiple differentiation culture experiments. The horizontal axis is the differentiation induction efficiency (%), and the vertical axis is the TRANCE concentration per unit cell number, and the data shows that the differentiation induction efficiency and the TRANCE concentration per unit cell number are correlated. FIG. 13 is a graph showing the correlation between the PDGA-AA concentration in the culture supernatant on differentiation induction Day 0 and the differentiation induction efficiency on differentiation induction Day 13 in cell groups with different differentiation efficiencies in multiple differentiation culture experiments. The horizontal axis is the differentiation induction efficiency (%), and the vertical axis is the PDGA-AA concentration per unit cell number, and the data shows that the differentiation induction efficiency and the PDGA-AA concentration per unit cell number are correlated. 14 is a graph showing the correlation between the FGF13 concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 in cell groups with different differentiation efficiencies in multiple differentiation culture experiments. The horizontal axis is the differentiation induction efficiency (%), and the vertical axis is the FGF13 concentration per unit cell number, showing the data indicating the correlation between the differentiation induction efficiency and the FGF13 concentration per unit cell number. FIG. 15 is a graph showing the correlation between the Kallikrein 6 concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 in cell groups with different differentiation efficiencies in multiple differentiation culture experiments. The horizontal axis is the differentiation induction efficiency (%), and the vertical axis is the Kallikrein 6 concentration per unit cell number, showing the data indicating the correlation between the differentiation induction efficiency and the Kallikrein 6 concentration per unit cell number. 16 is a graph showing the correlation between the MMP2 concentration in the culture supernatant on Day 0 of differentiation induction and the differentiation induction efficiency on Day 13 of differentiation induction in cell groups with different differentiation efficiencies in multiple differentiation culture experiments. The horizontal axis represents the differentiation induction efficiency (%), and the vertical axis represents the MMP2 concentration per unit cell number. The data show that the differentiation induction efficiency and the MMP2 concentration per unit cell number are correlated. FIG. 17A is a graph showing the amount of RAC2 present in the culture supernatant on Days 3, 6, and 12 after the start of differentiation induction in cell groups y1 and y2 with different differentiation efficiencies.The horizontal axis represents the number of days of culture after differentiation induction, and the vertical axis represents the peak area. The data show that immediately after the start of differentiation induction (Day 3), cell group y1 under conditions with high differentiation induction efficiency had a larger peak area. Figure 17B is a graph showing the time course of RAC2 concentration in the culture supernatant for cell groups z1 and z2 with different differentiation efficiencies. The horizontal axis represents the number of days of culture after differentiation induction, and the vertical axis represents the RAC2 concentration in the supernatant. The data show that immediately after the start of differentiation induction (Day 1), cell group z1 under conditions with high differentiation induction efficiency had a higher RAC2 concentration. Figure 17C is a graph showing the results of single-cell gene expression analysis of cell groups y1 and y2 with different differentiation efficiencies, plotted as a violin plot. The horizontal axis represents the number of days of culture after differentiation induction and the culture conditions, and the vertical axis represents counts per million (CPM), an index of average RNA expression level. 18A is a graph showing the amount of TKT present in the culture supernatant of cell groups y1 and y2 with different differentiation efficiencies on days 3, 6, and 12 after the start of differentiation induction. The horizontal axis represents the number of days of culture after differentiation induction, and the vertical axis represents the peak area. This data shows that immediately after the start of differentiation induction (Day 3), cell group y1 under conditions with high differentiation induction efficiency has a larger peak area. FIG. 18B is a graph showing the change in TKT concentration in the culture supernatant over time for cell groups z1 and z2 with different differentiation efficiencies. The horizontal axis represents the number of days of culture after differentiation induction, and the vertical axis represents the TKT concentration in the supernatant. This data shows that immediately after the start of differentiation induction (Day 1), cell group z1 under conditions with high differentiation induction efficiency has a higher TKT concentration. Figure 18C is a graph showing the results of single-cell gene expression analysis of cell groups y1 and y2 with different differentiation efficiencies in a violin plot. 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 indicator of average RNA expression level. The data show that at the start of differentiation (Day 0), cell group y1 under conditions with high differentiation induction efficiency had a higher average RNA amount of the TKT gene. Figure 19A is a graph showing the results of single-cell gene expression analysis of cell groups y1 and y2 with different differentiation efficiencies in a violin plot.The horizontal axis represents the number of culture days and culture conditions for differentiation induction, and the vertical axis represents counts per million (CPM), an index of average RNA expression level. This data shows that under conditions with high differentiation induction efficiency (y1), many cells exhibit high NPTX1 gene expression at the early stage of differentiation. Figure 19B is a graph showing the results of ELISA quantification of the time course of NPTX1 concentration in the culture supernatant for cell groups y1 and y2, and z1 and z2, with different differentiation efficiencies. Figure 19B shows the results of two experiments (Day 3 and Day 6 are for one experiment). The horizontal axis represents the number of culture days after differentiation induction, and the vertical axis represents the NPTX1 concentration in the supernatant. This data shows that immediately after the start of differentiation induction (day 1), the NPTX1 concentration was higher under conditions with high differentiation induction efficiency (y1, z1) than under conditions with low differentiation induction efficiency (y2, z2). 20 is a graph showing the change in NT-3 concentration in the culture supernatant over time for cell groups x1 and x2, which have different differentiation efficiencies. 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 with high differentiation induction efficiency (cell group x1).

[0024] 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.

[0025] <<Method (1) for Non-invasively Monitoring the State 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 non-invasively monitoring the state 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 non-invasively monitoring the state 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 a cell-secreted factor in a culture supernatant of 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 measured concentration of the cell-secreted factor 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 concentration of the cell-secreted factor is equal to or greater than the reference concentration, wherein the culture supernatant is collected from the culture medium at any time within 336 hours after the initiation of culturing the pluripotent stem cells. This is described in detail below.

[0026] <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.

[0027] (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).

[0028] 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.

[0029] 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.

[0030] 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 E-cadherin 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).

[0031] (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).

[0032] (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).

[0033] (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).

[0034] 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.

[0035] 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.

[0036] 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).

[0037] 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).

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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).

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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)).

[0046] 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).

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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, and 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.

[0051] 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.

[0052] (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.

[0053] (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.

[0054] <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.

[0055] <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 neural progenitor cells herein is preferably a cell population that does not contain serotonergic neural cells.

[0056] 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.

[0057] <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.

[0058] <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.

[0059] 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.

[0060] <Step of measuring the concentration of cell-secreted factors> In this step, the concentration of cell-secreted factors 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. In one aspect of this embodiment, the step of measuring the concentration of cell-secreted factors may include multiple measurements of the concentration of one or more cell-secreted factors that are expected to appear within 336 hours after the start of culture.

[0061] The present inventors selected candidate monitoring factors from proteins secreted into the culture supernatant through comprehensive gene expression analysis, proteome analysis, and literature analysis. From the selected candidate proteins, they then identified protein factors whose secretion levels showed a positive correlation with differentiation efficiency (sometimes referred to as "differentiation induction efficiency") using the Luminex method, single-cell gene expression analysis, and other methods. In the present invention, these protein factors are defined as "cell-secreted factors."That is, the cell-secreted factors according to this embodiment include Gas6 (growth arrest specific 6), SerpinF1 (serpin family F member 1), TFF3 (trefoil factor 3), WFDC2 (WAP four-disulfide core domain 2) (HE4: human epididymis protein 4), Cripto (TDGF1: teratocarcinoma-derived growth factor 1), CD30 (TNFRSF8: TNF receptor superfamily member 8), and B2M. (beta-2-microglobulin), CXCL6 (C-X-C motif chemokine ligand 6), IGFBP6 (insulin like growth factor binding protein) 6), HGFR (hepatocyte growth factor receptor) (MET: MET proto-oncogene, receptor tyrosine kinase), SPARC (secreted protein acidic and cysteine ​​rich), TRANCE (TNF-related Activation-induced Cytokine) (TNFSF11: TNF superfamily member 11), PDGF-AA (platelet derived growth factor-AA), FGF13 (fibroblast growth factor 13), Kalikrein6 (KLK6: Kalrikrein related peptide 6), MMP2 (matrix metallopeptidase 2), RAC2 (Rac family small GTPase 2), TKT (transketolase), NPTX1 (neuronal pentraxin 1) and NT-3 (NTF3: neurotrophin 3).In one aspect of this embodiment, the cell-secreted factor may be any one or more selected from the group consisting of Gas6, SerpinF1, TFF3, WFDC2, Cripto, CD30, B2M, CXCL6, IGFBP6, HGFR, SPARC, TRANCE, PDGF-AA, FGF13, Kallikrein6, MMP2, RAC2, TKT, NPTX1, and NT-3. Furthermore, the cell-secreted factor expected to appear within 336 hours after the initiation of culture may be any one or more selected from the group consisting of Gas6, SerpinF1, TFF3, WFDC2, Cripto, CD30, B2M, CXCL6, IGFBP6, HGFR, SPARC, TRANCE, PDGF-AA, FGF13, Kallikrein6, MMP2, RAC2, TKT, NPTX1, and NT-3. iPS cells with different genetic backgrounds may exhibit different rates of change in the cell-secreted factor during the differentiation process. For this reason, the concentration of the cell-secreted factor may be preliminarily measured in a medium containing an inducer of differentiation into neural cells of the midbrain floor plate region for each iPS cell with a different genetic background, and a correlation between the concentration of the cell-secreted factor and the efficiency of differentiation into the neural cells may be confirmed, thereby selecting an appropriate cell-secreted factor. Based on the concentration of the selected cell-secreted factor, the differentiation potential of the corresponding iPS cells into neural cells of the midbrain floor plate region may be determined.

[0062] (Concentration of Cell-Secreted Factor) The method for measuring the concentration of the cell-secreted factor 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, proximity extension assay, etc. An example of the multiplex suspension array system is Luminex (registered trademark) manufactured by Genetic Lab Co., Ltd.

[0063] In one aspect of this embodiment, the concentration of the cell-secreted factor in the culture supernatant may be measured using a highly sensitive measurement method. By using a highly sensitive measurement method, changes in the concentration of the cell-secreted factor in a low concentration range (e.g., 5 pg / ml to 20 pg / ml) can be detected sensitively and early. Examples of such highly sensitive measurement methods include digital ELISA and ProQuantum High Sensitivity Immunoassay (ProQuantum High Sensitivity Immunoassay, Thermo Fisher Scientific).

[0064] In one aspect of this embodiment, the method for measuring the concentration of the cell-secreted factor 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 the concentration of the cell-secreted factor or a method for monitoring both the increase in the concentration of the cell-secreted factor and the decrease in the concentration of the cell-secreted factor may be selected, and the change in the concentration of the cell-secreted factor during the production process may be measured in advance, and a method suitable for each production process may be adopted.

[0065] The culture supernatant is collected from the culture medium at any time point within 336 hours (e.g., 48 to 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"). Preferably, the culture supernatant is collected from the culture medium at any time point between 48 and 192 hours after the initiation of culture of the pluripotent stem cells. Here, "any time point within 336 hours after the initiation of culture" is not particularly limited and can be arbitrarily selected as long as a cell-secreted factor can be detected in the culture supernatant when differentiation into midbrain floor plate region neural cells proceeds normally. In other words, the timing of collection of the culture supernatant from the culture medium can be appropriately determined depending on the method for producing the target cells, such as midbrain floor plate region neural cells or dopaminergic neural progenitor cells, based on whether a cell-secreted factor concentration equal to or greater than the reference concentration can be detected at any time point within 336 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. In one aspect of this embodiment, the addition of the differentiation inducer to the medium used for culturing (e.g., expansion) pluripotent stem cells may be considered to be the start of "culturing in a medium containing a differentiation inducer for pluripotent stem cells."

[0066] The secretion levels of Gas6, RAC2, TKT, NPTX1, and NT-3 tend to peak between 24 and 96 hours after the start of pluripotent stem cell culture (i.e., after the start of differentiation induction). Therefore, when the cell-secreted factor to be monitored is Gas6, RAC2, TKT, NPTX1, or NT-3, the culture supernatant is preferably collected from the culture medium at any time within 216 hours after the start of pluripotent stem cell culture (preferably at any time between 24 and 96 hours after the start of culture).

[0067] The secretion amounts of SerpinF1 and TFF3 tend to increase 72 hours or more after the start of pluripotent stem cell culture (i.e., after the start of differentiation induction). Therefore, when the cell-secreted factor to be monitored is SerpinF1 or TFF3, the culture supernatant is preferably collected from the culture medium 72 to 312 hours after the start of pluripotent stem cell culture (preferably at any time between 120 and 312 hours after the start of culture).

[0068] CD30, WFDC2, and Cripto tend to be secreted before the initiation of pluripotent stem cell culture (i.e., before the initiation of differentiation induction), and the secretion level tends to peak between 24 and 72 hours after the initiation of the culture. Therefore, when the cell-secreted factor to be monitored is CD30, WFDC2, or Cripto, the culture supernatant is preferably a culture supernatant collected from the culture medium at any time point within 312 hours after the initiation of the pluripotent stem cell culture (preferably at any time point between 24 and 72 hours after the initiation of the culture).

[0069] B2M tends to be secreted before the start of pluripotent stem cell culture (i.e., before the start of differentiation induction), and the secretion amount tends to peak after 144 hours from the start of the culture, and the difference in supernatant concentration between groups with high and low differentiation induction efficiency becomes large between immediately after the start of differentiation induction and 24 hours from the start of culture. Therefore, when the cell-secreted factor to be monitored is B2M, the culture supernatant is preferably a culture supernatant collected from the culture medium at any time point within 288 hours after the start of pluripotent stem cell culture (preferably at any time point within 48 hours from the start of culture).

[0070] CXCL6, IBFBP6, HGFR, SPARC, TRANCE, PDGF-AA, FGF13, Kallikrein 6, or MMP2 tends to be secreted before the start of pluripotent stem cell culture (i.e., before the start of differentiation induction), and the supernatant concentration per unit cell number immediately after the start of differentiation induction correlates with the differentiation induction efficiency. Therefore, when the cell-secreted factor to be monitored is CXCL6, IBFBP6, HGFR, SPARC, TRANCE, PDGF-AA, FGF13, Kallikrein 6, or MMP2, the culture supernatant is preferably collected from the culture medium at any time point between 48 hours before the start of pluripotent stem cell culture (i.e., 48 hours before the start of differentiation induction) and 312 hours after the start of culture (preferably at the start of culture).

[0071] As described above, the cell-secreted factors defined in the present application can be used to non-invasively monitor the state of cells in culture at any time point, from before the start of culture in a medium containing a differentiation inducer of pluripotent stem cells to immediately after the start of culture, and throughout the period from the start of culture to differentiation into neural cells of the midbrain floor plate region. If a cell-secreted factor expected to appear in the culture medium at a predetermined time before or after the start of culture in a medium containing a differentiation inducer of pluripotent stem cells is not detected in the culture medium at a reference concentration or above, it is predicted that the cell state is not as expected. One possibility in this case is a detection error, and in addition to the one cell-secreted factor in the culture supernatant at the predetermined time, the concentrations of other cell-secreted factors in the culture supernatant at the predetermined time may also be measured.

[0072] Another possibility is that the overall progression of the differentiation process of a cell population may be delayed or accelerated due to differences in culture equipment (culture medium, culture vessel, culture device, etc.) and culture conditions, resulting in the concentration of an expected cell-secreted factor in the culture supernatant falling below the detection limit. In this case, in addition to the one cell-secreted factor in the culture supernatant at the specified time, the concentrations of other cell-secreted factors in the culture supernatant at the specified time may be measured. For example, if the Gas6 concentration is measured in the culture supernatant 48 hours after the start of culture but is not detected, RAC2, which is expected to appear in the culture supernatant at the same time as Gas6, may be detected, and if it is found to be at or above the reference concentration, it may be determined that differentiation is possible. In other words, detecting multiple cell-secreted factors in a single culture supernatant can address detection errors and fluctuations in the state of cells during differentiation, allowing for more accurate determinations. The same applies to the "second method for monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region" and the "third method for monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region" described below.

[0073] A modified example of the first method of monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region according to this embodiment may include the steps of measuring the concentrations of multiple cell-secreted factors in the culture supernatant of the culture medium obtained by culturing the pluripotent stem cells in a medium containing a differentiation inducer for neural cells of the midbrain floor plate region, comparing the measured concentrations of the multiple cell-secreted factors with corresponding reference concentrations, and 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 at least one of the multiple cell-secreted factors is equal to or greater than the corresponding reference concentration.

[0074] Alternatively, the concentration of other cell-secreted factors in the culture supernatant at a predetermined time may be measured and the differentiation potential determined by comparing the concentrations of the cell-secreted factors. For example, differentiation potential may be determined when the Gas6 concentration and SerpinF1 concentration in the culture supernatant are in a predetermined ratio. The ratio of Gas6 concentration to SerpinF1 concentration 48 hours after the start of culture is considered to be higher than the ratio of Gas6 concentration to SerpinF1 concentration 336 hours after the start of culture. Detecting multiple cell-secreted factors in multiple culture supernatants can address detection errors and fluctuations in the state of cells during differentiation progression, allowing for more accurate determination. The same applies to the "Second Method for Monitoring the State of Cells in Culture During Differentiation of Pluripotent Stem Cells into Neural System Cells of the Midbrain Floor Plate Region" and the "Third Method for Monitoring the State of Cells in Culture During Differentiation of Pluripotent Stem Cells into Neural System Cells of the Midbrain Floor Plate Region" described below.

[0075] Another variation of the first method of monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region according to this embodiment may include the steps of measuring the concentrations of multiple cell-secreted factors in the culture supernatant of the culture medium obtained by culturing the pluripotent stem cells in a medium containing a differentiation inducer into neural cells of the midbrain floor plate region, comparing the measured concentrations of the multiple cell-secreted factors, and determining that the cells in the culture medium are capable of differentiating into neural cells of the midbrain floor plate region when the ratio of the concentrations of the multiple cell-secreted factors is a predetermined ratio.

[0076] After differentiation induction begins, medium exchange is performed regardless of whether or not the concentration of cell-secreted factors is measured in order to ensure appropriate differentiation induction in the cell production process. The frequency of medium exchange is not particularly limited as long as the medium pH can be maintained within an appropriate range and the differentiation inducer and nutrients necessary for the cells are appropriately replenished. Typically, medium exchange is performed approximately every 12 hours, 24 hours, 48 ​​hours, or 72 hours. Furthermore, the medium exchange method may involve replacing the entire volume of the medium, or a partial volume, such as 3 / 4, half, 1 / 3, or 1 / 4. Here, when culturing is continued using a medium consisting of the same components, medium exchange is generally performed in a consistent manner throughout the culture period. Therefore, the concentration of cell-secreted factors contained in the culture supernatant is affected by the frequency and method of medium exchange. Therefore, although the detected concentration of cell-secreted factors varies depending on the individual production process, the culture supernatant can be collected at the appropriate time. Furthermore, an appropriate reference concentration can be set for each production process. That is, when setting the reference concentration, it is desirable to synchronize it with the collection of culture supernatant performed in the manufacturing process, taking into account the medium replacement method and schedule. Furthermore, to adjust the reference concentration between lots of medium or between lots of cells used, the concentrations of soluble components contained in the medium, such as lactate, glucose, ammonia, oxygen, carbon dioxide, glutamine, and glutamic acid, may be measured, and the reference concentration may be adjusted based on the measured concentrations of the soluble components. The concentrations of the soluble components may also be considered to be used as internal standards. These soluble components may be measured by sampling the medium or continuously while culturing the cells. The reference concentration may also be adjusted based on the number of cells in the culture medium. In this case, the number of cells may be estimated based on the amount of glucose consumed or the amount of lactate produced.

[0077] It is desirable that the "concentration of cell-secreted factors" correlates with the amount of cell-secreted factors produced by cells over a certain period of time. For example, in the case of a concentration in a culture supernatant collected after a culture medium exchange in which the entire volume is exchanged over a certain period of time, the concentration of cell-secreted factors secreted from the previous medium exchange to the current medium exchange is measured, and the "concentration of cell-secreted factors" referred to herein is the "concentration of cell-secreted factors." Therefore, when a culture supernatant is collected during a culture medium exchange, the "concentration of cell-secreted factors" corresponds to the concentration of cell-secreted factors in the culture supernatant discarded during the medium exchange operation. For example, the "concentration of cell-secreted factors" in the waste culture supernatant from a culture medium exchange performed every 24 hours corresponds to the amount of cell-secreted factors 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 of this embodiment, the concentration of cell-secreted factors may be the amount of cell-secreted factors appearing in the culture medium per unit time. The unit time may be, for example, 24 hours.

[0078] In one embodiment, when a portion of the culture supernatant is sampled from a medium that has not undergone medium exchange, i.e., when the medium has not been exchanged at least once between two samplings, the cumulative amount of cell-secreted factors secreted by the cells contained in the culture medium is measured. Therefore, by calculating the difference in the concentration of the cell-secreted factor between the two samplings, the calculated concentration corresponds to the increase in the "concentration of the cell-secreted factor" from the first sampling to the second sampling.

[0079] 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 concentration of cell-secreted factors 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 concentration of cell-secreted factors uniform before collecting the culture supernatant.

[0080] 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.

[0081] 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'-thioglycerol. 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.

[0082] (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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] (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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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), which is a cell membrane-permeable phosphorylated peptide. peptide inhibitor), 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 readily 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] <Step of Comparing the Measured Concentration of a Cell-Secreted Factor with a Reference Concentration> In this step, the measured concentration of the cell-secreted factor is compared with a reference concentration. The "reference concentration" refers to the concentration of the cell-secreted factor (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 also be the concentration of the cell-secreted factor 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: 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. When target cells that achieve the goal are obtained, i.e., when the differentiation induction efficiency is good, the concentration of the cell-secreted factor in the culture supernatant is measured. Such culture and measurement can be performed multiple times (at least two times, preferably at least five times), and the average concentration of the cell-secreted factor measured in each test 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 concentration of the cell-secreted factor in the culture supernatant when target cells that achieve the target are obtained, and the concentration of the cell-secreted factor 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.

[0098] 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.

[0099] As used herein, "when target cells that achieve the target are not obtained" or "when the efficiency of differentiation induction is poor" refers to cases where target cells are not obtained in a quantity (proportion) that meets the target despite differentiation induction according to the manufacturing procedure (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 target quantity, at least twice, and preferably at least five times, and then set the average concentration of cell-secreted factors measured in each case as the "concentration of cell-secreted factors in the culture supernatant in which differentiation induction efficiency is poor." Alternatively, "when target cells that achieve the target are not obtained" or "when the efficiency of differentiation induction is poor" may be, for example, a case where culture is performed in a medium or culture conditions that deliberately 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 target quantity. 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 properly controlled in the manufacturing protocol. In this case, it is desirable to obtain "incompatibility" results, in which the target amount of target cells is not obtained, by differentiation induction that deviates from the specifications at least twice, and preferably at least five times, and to set the average concentration of cell-secreted factors measured in each case as the "concentration of cell-secreted factors in the culture supernatant when differentiation induction is inefficient." Here, "conditions that deviate from the specifications of the manufacturing protocol" refers to conditions under which differentiation induction into target cells proceeds appropriately but the 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."

[0100] 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.

[0101] Furthermore, the appropriate timing for detecting the concentration of cell-secreted factors can be appropriately set by culturing pluripotent stem cells in a medium and culture conditions that allow differentiation, 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 concentration of cell-secreted factors can be effectively measured. For example, culturing and measurement can be performed multiple times (e.g., twice, preferably five times) over time, and the timing at which the concentration of cell-secreted factors 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 concentration of the cell-secreted factors can be measured, and an appropriate measurement frequency can be set accordingly. For example, measurement can be performed every time the medium is replaced.

[0102] Specifically, the reference concentration is preferably set to a value between 0.1 pg / ml and 10,000 pg / ml, more preferably between 0.3 pg / ml and 3,000 pg / ml, more preferably between 0.3 pg / ml and 100 pg / ml. ６It may be set to pg / ml or less.

[0103] In one aspect of this embodiment, the reference concentration may be the concentration of the cell-secreted factor before the secretion amount of the cell-secreted factor increases. For example, the reference concentration may be the concentration of the cell-secreted factor measured within 48 hours, preferably within 24 hours, after the start of differentiation induction. In another aspect of this embodiment, the reference concentration may be the minimum concentration at which differentiation is successful.

[0104] In this embodiment, the "state of cells in the culture medium" refers to the state of cells in the culture medium, including their ability to differentiate into target cells (i.e., differentiation potential), the appropriate progress of differentiation, and the direction of differentiation. The "state of cells in the culture medium" is affected by the composition of the culture medium, the temperature of the culture medium, dissolved gases in the culture medium, the ratio of cells to the culture medium, etc.

[0105] <Step of Determining That Cells in a Culture Solution Can Differentiate into Midbrain Floor Plate Region Neural Cells> In this step, when the concentration of the cell-secreted factor 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 solution at the time the concentration of the cell-secreted factor 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 differentiation" can also be understood as "possibly capable of differentiation." In addition, in one aspect of this embodiment, "capable of differentiation" may also mean that a target amount of midbrain floor plate region neural cells can be expected to be obtained in the differentiation induction step in the production of midbrain floor plate region neural cells. In addition, in one aspect of this embodiment, "determining that cells can be differentiated" 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, when it is estimated that the target amount of neural cells of the midbrain floor plate region cannot be obtained in the differentiation induction step, this corresponds to a case where "it cannot be determined that they can be differentiated." Furthermore, the "concentration of cell-secreted factors" 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."

[0106] <Step of Determining the Presence or Absence of Abnormalities in Cells in the Culture Medium> As one aspect of the step of determining whether cells in the culture medium can differentiate into neural cells of the midbrain floor plate region, the concentration of a cell-secreted factor may be compared with a reference concentration to determine whether the differentiation process into neural cells of the midbrain floor plate region is within an acceptable range (no abnormalities in the cells). That is, the method according to this embodiment may further include a step of determining the presence or absence of abnormalities in the cells in the culture medium. A determination that the cells in the culture medium are abnormal is made by comparing the concentration of a cell-secreted factor with a reference concentration and determining that the differentiation process is within a normal range and that the quantity and quality of the target cells to be obtained after culture can be ensured to be sufficient (determining that the cells in the culture medium can differentiate into neural cells of the midbrain floor plate region), and determining that no events that would necessitate discontinuing the culture (e.g., contamination, detection of differentiation into non-target cells) exist.

[0107] On the other hand, a determination that cells in the culture medium are abnormal occurs when the concentration of cell-secreted factors is compared with a reference concentration and it is determined that the differentiation process is outside the normal range and that the quantity and quality of the target cells to be obtained after culture are insufficient (when it cannot be determined that differentiation is possible). Alternatively, it occurs when it is determined that the differentiation process is within the normal range but an event that necessitates interrupting the culture (contamination, detection of differentiation into non-target cells) has occurred. Events that necessitate interruption of the culture can be detected by general-purpose means such as inspecting the culture equipment, checking the color of the medium, observing the cells under a microscope, testing for microbial contamination, or invasive testing using collected cells.

[0108] The method of the present invention for non-invasively monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region (hereinafter, sometimes referred to as the "monitoring method of the present invention") allows early determination of 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 monitoring method of the present invention is extremely useful for managing the manufacturing process. Furthermore, the monitoring 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. It can also be used to determine the efficiency of differentiation induction when optimizing conditions for scale-up of manufacturing.

[0109] In one aspect of this embodiment, the phrase "the concentration of the cell-secreted factor is equal to or greater than the reference concentration" encompasses cases where the concentration of the cell-secreted factor is equal to the reference concentration and cases where the concentration of the cell-secreted factor is higher than the reference concentration. 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 concentration of the cell-secreted factor is 1 to 1000 times the reference concentration, and it is more preferable to determine that the cells contained in the culture medium at the time the concentration of the cell-secreted factor was measured are capable of differentiating into neural cells of the midbrain floor plate region when the concentration of the cell-secreted factor is 1.2 to 1000 times the reference concentration.

[0110] The percentage of a cell population determined by the monitoring method of the present invention 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, based on the total number of cells being cultured, 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 will differentiate into neural cells of the midbrain floor plate region.

[0111] In one aspect of this embodiment, from the viewpoint of efficient differentiation induction, when the concentration of the cell-secreted factor 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 should be adjusted appropriately 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.

[0112] <<Method (2) for Non-invasively Monitoring the State 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 for monitoring the state 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 non-invasively monitoring the state 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 cell-secreted factors 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 concentration of the cell-secreted factor from the concentration of the cell-secreted factor in the first culture supernatant and the concentration of the cell-secreted factor in the second culture supernatant; comparing the absolute value of the rate of change in the concentration of the cell-secreted factor 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 concentration of the cell-secreted factor is equal to or greater than the reference rate of change, wherein the first culture supernatant is collected from the culture medium at any time within 336 hours after the start of culturing the pluripotent stem cells, and the second culture supernatant is collected from the culture medium at any time between 24 and 72 hours before or between 24 and 72 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.

[0113] <Step of measuring the concentration of cell-secreted factors> In this step, the concentrations of cell-secreted factors 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 concentration measurement method can be the same as the measurement method mentioned in the first method above.

[0114] The first culture supernatant is a culture supernatant collected from the culture medium at any time within 336 hours after the initiation of culturing the pluripotent stem cells, and in one embodiment, may be a culture supernatant collected from the culture medium at any time between 48 and 96 hours after the initiation of culturing the pluripotent stem cells. In another embodiment, the first culture supernatant may be a culture supernatant collected from the culture medium at any time between 48 and 72 hours after the initiation of culturing the pluripotent stem cells.

[0115] 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 can be collected from the culture medium any time between 24 hours and 72 hours (preferably 24 to 48 hours) before the collection of the first culture supernatant. In another embodiment, the second culture supernatant is 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 any time between 24 hours after the collection of the first culture supernatant and before the completion of differentiation into neural cells of the midbrain floor plate region. 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 72 hours before collection of the first culture supernatant or at any time between 24 and 72 hours (more preferably, 24 and 48 hours) after collection of the first culture supernatant. Here, "any time between 24 and 72 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 72 hours before collection of the first culture supernatant.

[0116] In one aspect of this embodiment, when the cell-secreted factor to be monitored is Gas6, RAC2, TKT, NPTX1, or NT-3, it is preferable that the first culture supernatant is a culture supernatant collected from the culture medium at any time within 216 hours after the initiation of culturing the pluripotent stem cells (preferably any time between 24 and 96 hours after the initiation of culturing), and the second culture supernatant is a culture supernatant collected from the culture medium at any time between 24 and 72 hours before the collection of the first culture supernatant or at any time between 24 and 72 hours after the collection of the first culture supernatant.

[0117] In one aspect of this embodiment, when the cell-secreted factor to be monitored is SerpinF1 or TFF3, it is preferable that the first culture supernatant is a culture supernatant collected from the culture medium between 72 and 312 hours after the start of culturing the pluripotent stem cells (preferably at any time between 120 and 312 hours after the start of culturing), and the second culture supernatant is a culture supernatant collected from the culture medium between 24 and 72 hours before the collection of the first culture supernatant.

[0118] In one aspect of this embodiment, when the cell-secreted factor to be monitored is CD30, WFDC2, or Cripto, it is preferable that the first culture supernatant is a culture supernatant collected from the culture medium at any time within 312 hours after the start of culturing the pluripotent stem cells (preferably at any time between 24 and 72 hours after the start of culturing), and the second culture supernatant is a culture supernatant collected from the culture medium at any time between 24 and 72 hours before the collection of the first culture supernatant or at any time between 24 and 72 hours after the collection of the first culture supernatant.

[0119] In one aspect of this embodiment, when the cell-secreted factor to be monitored is B2M, the first culture supernatant is preferably a culture supernatant collected from the culture medium at any time within 288 hours after the start of culturing the pluripotent stem cells (preferably any time within 48 hours after the start of culturing), and the second culture supernatant is preferably a culture supernatant collected from the culture medium at any time between 24 and 72 hours before the collection of the first culture supernatant or at any time between 24 and 72 hours after the collection of the first culture supernatant.

[0120] In one aspect of this embodiment, when the cell-secreted factor to be monitored is CXCL6, IBFBP6, HGFR, SPARC, TRANCE, PDGF-AA, FGF13, Kallikrein 6, or MMP2, it is preferable that the first culture supernatant is a culture supernatant collected from the culture medium at any time point between 48 hours before the start of culturing the pluripotent stem cells (i.e., 48 hours before the start of differentiation induction) and within 312 hours after the start of culturing (preferably at the start of differentiation induction), and the second culture supernatant is a culture supernatant collected from the culture medium at any time point between 24 hours and 72 hours before the collection of the first culture supernatant, or at any time point between 24 hours and 72 hours after the collection of the first culture supernatant.

[0121] The composition of the medium for culturing the pluripotent stem cells can be the same as that of the first method described above.

[0122] <Step of determining the rate of change in the concentration of cell-secreted factor> In this step, the rate of change in the concentration of cell-secreted factor is determined from the concentration of the cell-secreted factor in the first culture supernatant and the concentration of the cell-secreted factor in the second culture supernatant. The rate of change in the concentration of cell-secreted factor can be determined using the following formula: When the second culture supernatant is collected before the first culture supernatant: (rate of change in the concentration of cell-secreted factor) = {(concentration of cell-secreted factor in the first culture supernatant) - (concentration of cell-secreted factor 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 the concentration of cell-secreted factor) = {(concentration of cell-secreted factor in the second culture supernatant) - (concentration of cell-secreted factor in the first culture supernatant)} / {(time when the second culture supernatant is collected) - (time when the first culture supernatant is collected)}

[0123] In one aspect of this embodiment, the concentration of the cell-secreted factor in the culture supernatant may temporarily increase and decrease over 24 or 48 hours during the period from the initiation of differentiation to day 14 (the period from the initiation of culture of the pluripotent stem cells to 336 hours after the initiation of culture). In another aspect of this embodiment, the concentration of the cell-secreted factor in the culture supernatant may temporarily increase during the period from the initiation of differentiation to day 14, and the increased period may last for approximately 168 hours. Here, the "increased period" refers to the period during which the concentration of the cell-secreted factor is higher than the baseline value. As long as the concentration is higher than the baseline value, the "increased period" includes even if the concentration of the cell-secreted factor remains constant or decreases temporarily. In one aspect of this embodiment, the concentration of the cell-secreted factor in the culture supernatant may continuously increase during the period from the initiation of differentiation to day 14 (the period from the initiation of culture of the pluripotent stem cells to 336 hours after the initiation of culture). The timing of collecting the first culture supernatant and the second culture supernatant can be appropriately determined depending on the characteristics of the changes in the concentration of the cell-secreted factor in each differentiation induction step.

[0124] <Step of Comparing the Absolute Value of the Rate of Change in the Concentration of a Cell-Secreted Factor with a Reference Rate of Change> In this step, the absolute value of the rate of change in the concentration of the cell-secreted factor 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 the concentration of a cell-secreted factor that serves as a reference 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 the concentration of a cell-secreted factor determined when pluripotent stem cells known to differentiate into midbrain floor plate region neural cells are cultured under specified conditions, or it may be the absolute value of a preset rate of change. The preset absolute value of the rate of change can be set, for example, as follows: First, pluripotent stem cells are cultured in a medium and culture conditions capable of inducing differentiation, i.e., according to the production procedure (protocol) for midbrain floor plate region neural cells, and the "absolute value of the rate of change in the concentration of a cell-secreted factor" 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 in the concentration of the cell-secreted factor" determined for each culture can be set as the reference rate of change. Alternatively, when pluripotent stem cells are cultured in a differentiation-inducing medium and under culture conditions, the "absolute value of the rate of change in the concentration of the cell-secreted factor" when a target cell that achieves the target is obtained and the "absolute value of the rate of change in the concentration of the cell-secreted factor" when a target cell that achieves the target is not obtained, i.e., when the efficiency of differentiation induction is poor, are used to estimate the relationship between the reference rate of change and the sensitivity and specificity. Here, the meanings of "when a target cell that achieves the target is obtained" and "when differentiation induction efficiency is good," as well as "when a target cell that achieves the target is not obtained" and "when differentiation induction efficiency is poor," are the same as those described in the section "Step of comparing the measured concentration of the cell-secreted factor with the reference concentration" in the first method.

[0125] 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 the concentration of the cell-secreted factor in the first culture supernatant is detected as being higher than the concentration of the cell-secreted factor in the second culture supernatant. In this case, the above-mentioned rate of change may be calculated, or the determination may be made by determining that the concentration of the cell-secreted factor in the first culture supernatant is higher than the concentration of the cell-secreted factor in the second culture supernatant. In another 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 the concentration of the cell-secreted factor in the second culture supernatant is detected as being lower than the concentration of the cell-secreted factor in the first culture supernatant. In this case, the above-mentioned rate of change may be calculated, or the determination may be made by determining that the concentration of the cell-secreted factor in the second culture supernatant is lower than the concentration of the cell-secreted factor in the first culture supernatant.

[0126] The above-mentioned standard rate of change is 0.1 pg / ml / day or more. ６ It is preferable to set it at one point between 1000 and 1000 pg / ml·day or less.

[0127] <Step of Determining the Presence or Absence of Abnormalities in Cells in the Culture Solution> As one aspect of the step of determining whether cells in the culture solution can differentiate into neural system cells of the midbrain floor plate region, the absolute value of the rate of change in the concentration of a cell-secreted factor may be compared with a reference rate of change to determine whether the differentiation process into neural system cells of the midbrain floor plate region is within an acceptable range (no abnormalities in the cells). That is, the method according to this embodiment may further include a step of determining whether or not the cells in the culture solution are abnormal. Determining that cells in the culture solution are abnormal means comparing the absolute value of the rate of change in the concentration of a cell-secreted factor with a reference rate of change to determine that the differentiation process is within a normal range and that the quantity and quality of the target cells to be obtained after culture can be ensured (determining that the cells in the culture solution can differentiate into neural system cells of the midbrain floor plate region), and determining that no events that would necessitate interruption of the culture (e.g., contamination, detection of differentiation into non-target cells) exist.

[0128] On the other hand, a determination that cells in the culture medium are abnormal occurs when the absolute value of the rate of change in the concentration of cell-secreted factors is compared with a reference rate of change and it is determined that the differentiation process is outside the normal range and that the quantity and quality of the target cells to be obtained after culture are insufficient (when it cannot be determined that differentiation is possible). Alternatively, it is determined that the differentiation process is within the normal range, but an event that necessitates interrupting the culture (contamination, detection of differentiation into non-target cells) has occurred. Events that necessitate interruption of the culture can be detected by general-purpose means such as inspecting the culture equipment, checking the color of the medium, observing the cells under a microscope, testing for microbial contamination, or invasive testing using collected cells.

[0129] In one aspect of this embodiment, the phrase "the absolute value of the rate of change in the concentration of the cell-secreted factor is equal to or greater than the reference rate of change" encompasses cases where the absolute value of the rate of change in the concentration of the cell-secreted factor is equal to the reference rate of change and cases where the absolute value of the rate of change in the concentration of the cell-secreted factor 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 the concentration of the cell-secreted factor 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 the concentration of the cell-secreted factor is 1 to 1,000 times the reference rate of change.

[0130] 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.

[0131] In one aspect of this embodiment, from the viewpoint of efficient differentiation induction, when the absolute value of the rate of change in the concentration of the cell-secreted factor 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.

[0132] A variation of the second method of monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region according to this embodiment may include the steps of: measuring the concentrations of multiple cell-secreted factors 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 a differentiation inducer for neural cells of the midbrain floor plate region; determining the rate of change in the concentrations of the multiple cell-secreted factors from the concentrations of the multiple cell-secreted factors in the first culture supernatant and the second culture supernatant; comparing the absolute value of the rate of change in the concentrations of the multiple cell-secreted factors with a corresponding standard 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 concentration of at least one of the multiple cell-secreted factors is equal to or greater than the corresponding standard rate of change.

[0133] <<Method (3) for Non-invasively Monitoring the State 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 non-invasively monitoring the state 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 non-invasively monitoring the state 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 cell-secreted factors 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 the cell-secreted factors 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 the cell-secreted factor in the first culture supernatant is higher than the concentrations of the cell-secreted factor in the second culture supernatant and the third culture supernatant, wherein the first culture supernatant is collected from the culture medium at any time within 336 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 72 hours before the collection of the first culture supernatant, and the third culture supernatant is collected from the culture medium at any time between 24 hours and 72 hours after the collection of the first culture supernatant.

[0134] <Step of measuring the concentration of cell-secreted factors> In this step, the concentrations of cell-secreted factors are measured in the first culture supernatant, the second culture supernatant, and the third culture supernatant in the 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. The concentration measurement method can be the same as the measurement method mentioned in the first method above.

[0135] 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.

[0136] 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 72 hours (preferably 24 to 48 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 any time between 24 and 72 hours (more preferably, 24 to 48 hours) before the collection of the first culture supernatant, and is preferably collected from the culture medium 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 as to detect whether the concentration of the cell-secreted factor in the second culture supernatant is higher than the concentration of the cell-secreted factor in the first culture supernatant.

[0137] The third culture supernatant is collected from the culture medium at any time between 24 hours and 72 hours after collection of the first culture supernatant until differentiation into midbrain floor plate region neural cells is complete. The third culture supernatant is preferably collected from the culture medium at any time between 24 hours and 72 hours after collection of the first culture supernatant, and more preferably collected from the culture medium at any time between 24 hours and 48 hours after collection of the first culture supernatant. In one aspect of this embodiment, the timing of collection of each culture supernatant may be determined so that the concentration of cell-secreted factors in the third culture supernatant is detected to be lower than the concentration of cell-secreted factors in the first culture supernatant. Here, "completion of differentiation into midbrain floor plate region neural cells" refers to a state in which cell differentiation has progressed to the point where markers for midbrain floor plate region neural cells are expressed. In this case, the culture medium may contain neural cells that have further differentiated from midbrain floor plate region neural cells. 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.

[0138] In one aspect of this embodiment, when the cell-secreted factor to be monitored is Gas6, RAC2, TKT, NPTX1, or NT-3, it is preferable that the first culture supernatant is a culture supernatant collected from the culture medium at any time within 216 hours after the initiation of culturing the pluripotent stem cells (preferably any time between 24 and 96 hours after the initiation of culturing), the second culture supernatant is a culture supernatant collected from the culture medium at any time between 24 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 at any time between 24 and 72 hours after the collection of the first culture supernatant.

[0139] In one aspect of this embodiment, when the cell-secreted factor to be monitored is CD30, WFDC2, or Cripto, it is preferable that the first culture supernatant is a culture supernatant collected from the culture medium at any time within 312 hours after the start of culturing the pluripotent stem cells (preferably at any time between 24 and 72 hours after the start of culturing), the second culture supernatant is a culture supernatant collected from the culture medium at any time between 24 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 at any time between 24 and 72 hours after the collection of the first culture supernatant.

[0140] The composition of the medium for culturing the pluripotent stem cells can be the same as that of the first method described above.

[0141] <Step of comparing the concentrations of cell-secreted factors> In this step, the concentrations of the cell-secreted factors in the first culture supernatant, the second culture supernatant, and the third culture supernatant are compared.

[0142] <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 the cell-secreted factor in the first culture supernatant is higher than the concentration of the cell-secreted factor in the second culture supernatant and the concentration of the cell-secreted factor 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.

[0143] In one aspect of this embodiment, when the difference in concentration between the cell-secreted factor in the first culture supernatant and the lower of the concentrations of the cell-secreted factor in each of the second and third culture supernatants 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 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 difference in concentration is 10 pg / ml or more.

[0144] 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.

[0145] <Step of Determining the Presence or Absence of Abnormalities in Cells in the Culture Solution> As one aspect of the step of determining whether cells in the culture solution can differentiate into neural cells of the midbrain floor plate region, the concentrations of the cell-secreted factors in the first culture supernatant, the second culture supernatant, and the third culture supernatant may be compared to determine whether the differentiation process into neural cells of the midbrain floor plate region is within an acceptable range (no cell abnormalities). That is, the method according to this embodiment may further include a step of determining the presence or absence of abnormalities in cells in the culture solution. When determining that there are no abnormalities in the cells in the culture medium, the concentrations of the cell-secreted factors in the first culture supernatant, the second culture supernatant, and the third culture supernatant are compared to determine that the differentiation process is within a normal range and that the quantity and quality of the target cells to be obtained after culture can be ensured to be sufficient (it is determined that the cells in the culture medium can differentiate into neural cells in the midbrain floor plate region), and it is determined that there are no events that would require the culture to be discontinued (such as contamination or detection of differentiation into non-target cells).

[0146] On the other hand, the presence of an abnormality in the cells in the culture medium is determined by comparing the concentrations of the cell-secreted factors in the first culture supernatant, the second culture supernatant, and the third culture supernatant, and determining that the differentiation process is outside the normal range and that the quantity and quality of the target cells to be obtained after culture are insufficient (when it cannot be determined that differentiation is possible). Alternatively, the differentiation process is determined to be within the normal range, but an event that necessitates interruption of culture (contamination, detection of differentiation into non-target cells) has occurred. Events that necessitate interruption of culture may be detected by commonly used means, such as inspection of the culture apparatus, confirmation of the color of the medium, microscopic observation of the cells, testing for microbial contamination, or invasive testing using collected cells.

[0147] In one aspect of this embodiment, from the viewpoint of efficient differentiation induction, when the concentration of the cell-secreted factor in the first culture supernatant is the same as or lower than the concentrations of the cell-secreted factor 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.

[0148] A variation of the third method of monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region according to this embodiment may include the steps of: measuring the concentrations of multiple cell-secreted factors in each of 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 cells of the midbrain floor plate region; comparing the concentrations of the multiple cell-secreted factors 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 cells of the midbrain floor plate region when the concentration of at least one of the multiple cell-secreted factors in the first culture supernatant is higher than the concentrations of the multiple cell-secreted factors in the second culture supernatant and the third culture supernatant.

[0149] <Monitoring method using multiple factors> In one embodiment, the monitoring method of the present invention can be carried out by combining, in addition to the cell-secreted factors described above, one or more substances other than the cell-secreted factors that are detected in the culture supernatant during the process of differentiation into neural cells in the midbrain floor plate region as indicators for assessment.

[0150] The "one or more substances other than cell-secreted factors" are 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 monitoring method of the present invention by univariate logistic regression analysis, for example.

[0151] When the monitoring method of the present invention is performed by combining the above-mentioned cell-secreted factors with "one or more substances other than cell-secreted factors," a model formula optimal for determining the differentiation ability of the midbrain floor plate region into nervous system cells in the above-mentioned combination may be determined in advance using multivariate logistic regression analysis, Cox regression analysis, or the like, and the differentiation ability of the midbrain floor plate region into nervous system cells may be determined based on the model formula thus determined.

[0152] <Monitoring Method Using a Culture Device> In one aspect of this embodiment, the monitoring method can be used in a culture device for inducing differentiation of pluripotent stem cells. Examples of the culture device include a culture vessel for culturing target cells 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 above-mentioned methods (1) to (3) for non-invasively monitoring the state 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 the secreted factors contained in the discharged liquid medium, and a differentiation potential assessment unit for determining whether the target cells can differentiate into midbrain floor plate region neural cells based on the measured concentration of the secreted factors. 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 monitoring 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 the secreted factors contained in the waste liquid collected during medium exchange may be incorporated into the culture device, allowing only batches with the potential to differentiate into midbrain floor plate region neural cells to be selected for continued culture.

[0153] 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.

[0154] The above describes a method for non-invasively monitoring the state 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 monitoring method is useful in established methods for producing dopaminergic neural progenitor cells, which are the active ingredients of cellular pharmaceuticals and are obtained by inducing differentiation from pluripotent stem cells. When implementing this monitoring method, it is desirable to first perform a method for producing dopaminergic neural progenitor cells to understand the secretion pattern of cell-secreted factors, and then determine the optimal timing for measuring the secretion amount of cell-secreted factors for each production method. This monitoring method can be suitably applied to at least cells in the midbrain floor plate region (also known as midbrain floor plate cells), i.e., Corin-positive cells, that can produce a certain number of Corin-positive cells.

[0155] The monitoring method described above 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 monitoring method can be suitably applied as long as usable iPS cells that have been cultured under appropriate culture conditions are used.

[0156] <<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 capable of inducing differentiation into neural system cells of the midbrain floor plate region; (B) a step of non-invasively monitoring the state of cells in the culture solution during the differentiation of the pluripotent stem cells into neural system cells of the midbrain floor plate region between 0 and 336 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 solution 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 solution 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.

[0157] <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.

[0158] (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.

[0159] 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.

[0160] 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.

[0161] 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:

[0162] 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.

[0163] <Step (B): Performing a method for non-invasively monitoring the state 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 non-invasively monitoring the state 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 336 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 non-invasively monitoring the state 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 336 hours after the start of culturing the pluripotent stem cells. In one aspect of this embodiment, "between 0 hours and 336 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."

[0164] 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).

[0165] <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).

[0166] 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).

[0167] 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.

[0168] 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.

[0169] 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 process: (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.

[0170] 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).

[0171] 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.

[0172] 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.

[0173] 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.

[0174] <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.

[0175] 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.

[0176] <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.

[0177] The culture conditions (medium, temperature, carbon dioxide concentration, etc.) that allow differentiation into dopaminergic neural progenitor cells are described below.

[0178] 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, and 3'-thioglycerol. Alternatively, the medium may 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, and nucleic acids (e.g., dibutylyl cyclic AMP (dbcAMP)). 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.

[0179] <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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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 (trademark)) having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit).

[0188] In one aspect of this embodiment, step (D) is preferably 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] Methods for detecting target cells, such as midbrain floor plate cells, include the use of a flow cytometer or a protein chip.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] From the viewpoint of preventing rejection reactions, it is preferable that the HLA genotype of the cells contained in the tissue for transplantation is the same or substantially the same as the HLA genotype of the individual receiving the transplant.

[0199] 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).

[0200] Examples of the present invention will be described below, but the present invention is not limited to these. Differentiation of iPS cells that maintain an undifferentiated state into neural cells in the midbrain floor plate region was induced. At various times after the start of differentiation induction, we measured the concentration of cell-secreted factors in the culture supernatant, measured the amount of proteins present in the culture supernatant by proteome analysis, and measured gene expression by single-cell gene expression analysis. These results revealed the presence of cell-secreted factors (Gas6, SerpinF1, TFF3, WFDC2, Cripto (TDGF1), CD30, B2M, CXCL6, IGFBP6, HGFR, SPARC, TRANCE, PDGF-AA, FGF13, Kallikrein6, MMP2, RAC2, TKT, NPTX1, NT-3, PCSK1 (proprotein convertase subtilisin / kexin type 1), ENPP2 (ectonucleotide The relationship between the expression of α-pyrophosphatase / phosphodiesterase 2 (PARP1), poly(ADP-ribose) polymerase 1 (PARP1), DX39B (DDX39B: DExD-box helicase 39B), and AMD (PAM: peptidylglycine alpha-amidating monooxygenase) and cell differentiation efficiency was investigated.

[0201] <<Culture for maintaining undifferentiated state>> Prior to the differentiation-inducing culture into neural cells in the midbrain floor plate region, described below, iPS cells were cultured while maintaining their undifferentiated state. The iPS cell lines used were the 201B7 line, the 1231A3 line, and the S line, which was established from adult peripheral blood mononuclear cells using a Sendai virus vector (CytoTune™-2.0; ID Pharma).

[0202] <<Culture for Inducing 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 multiple cell types prepared in Culture Experiments 1 to 5 described below. The differentiation-inducing medium used was Glasgow's MEM supplemented with 8% (v / v) Knockout Serum Replacement, 1% (v / v) Non-Essential Amino Acids Solution, 1 mM Sodium Pyruvate, and 0.1 mM 2-mercaptoethanol. On day 0 after the start of differentiation, 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 the start of differentiation, Purmorphamine (an SHH signaling activator, final concentration 2 μM) and FGF8 (final concentration 100 ng / mL) were added to the medium. Three days after the start of differentiation, CHIR99021 (a Wnt signaling activator, final concentration 3 μM) was added. Seven days after the start of differentiation, the addition of A-83-01, Purmorphamine, and FGF8 was terminated. From the start of differentiation to day 13, the entire medium was replaced every 24 hours. 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 day 12 or 13 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:Glasgow's MEM: manufactured by Thermo Fisher; Knockout Serum Replacement: manufactured by Thermo Fisher; Non-Essential Amino Acids Solution: manufactured by Thermo Fisher; Sodium Pyruvate: manufactured by Thermo Fisher; 2-Mercaptoethanol: manufactured by Wako; A-83-01: manufactured by Wako; LDN-193189: manufactured by STEMGENT; Purmorphamine: manufactured by Wako; FGF8: manufactured by Wako; CHIR99021: manufactured by Wako.

[0203] <Culture Experiments> iPS cells were cultured to induce differentiation into neural cells of the midbrain floor plate region. Five culture experiments were conducted under the following conditions. (Culture Experiment 1) iPS cell line 201B7 was used to maintain undifferentiated cells and to induce differentiation into neural cells of the midbrain floor plate region. Cell groups x1 and x2 were established using different medium components. (Culture Experiment 2) iPS cell line 201B7 was used to maintain undifferentiated cells and to induce differentiation into neural cells of the midbrain floor plate region. Cell groups y1 and y2 were established using different medium components. (Culture Experiment 3) iPS cell line 201B7 was used to maintain undifferentiated cells and to induce differentiation into neural cells of the midbrain floor plate region. Cell groups z1 and z2 were established using different medium components. (Culture Experiment 4) iPS cell line S was used to maintain undifferentiated cells and to induce differentiation into neural cells of the midbrain floor plate region three times, and multiple culture supernatants were obtained. (Culture Experiment 5) iPS cell line 1231A3 was used to maintain undifferentiated cells in culture and induce differentiation into neural cells in the midbrain floor plate region. SAG (N-Methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane, final concentration 300 nM) was added to the culture medium 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, w1, was established. For comparison, a group with low induction efficiency, w2, was established by culturing cells under the same conditions except that SAG was not added to the culture medium.

[0204] <Evaluation of Differentiation Induction Efficiency> In each of the five culture experiments, the Corin positivity rate on day 12 or 13 after the start of differentiation was used as an indicator of differentiation induction efficiency. A portion of the cells from each cell group was collected on day 12 or 13 after the start of differentiation, and the percentage of Corin-positive cells in each cell group was measured using a cell sorter (BD FACSJazz, BD Accuri, or Furukawa PERFLOW Sort), which was used as the Corin positivity rate. The Corin positivity rates in each culture experiment were as follows: (Culture Experiment 1) 83% for cell group x1 and 24% for cell group x2. (Culture Experiment 2) 58.2% for cell group y1 and 34.1% for cell group y2. (Culture Experiment 3) 66.5% for cell group z1 and 56.9% for cell group z2. (Culture experiment 4) The results were 75.0%, 41.5%, and 70.9% in three culture experiments, respectively. (Culture experiment 5) The results were 90% for cell group w1 and 41% for cell group w2.

[0205] <<Obtaining Culture Supernatant>> A portion of the culture supernatant from each cell group was collected every 24 hours or every 48 hours during the undifferentiated maintenance culture period (expansion culture period) and differentiation induction culture period, and the concentration of candidate proteins for cell-secreted factors was analyzed. The timing of collection of the culture supernatant was set at a predetermined time within 24 hours (1 day) to coincide with the timing of medium change. 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 well stirred.

[0206] <<Analysis of Cell-Secreted Factor Concentration>> Immediately after the initiation of differentiation-inducing culture (i.e., immediately after the initiation of differentiation), a portion of the culture supernatant from each cell group was collected every 24 hours or 48 hours, and the concentration of cell-secreted factors was analyzed. The timing of collection of the culture supernatant was set at a predetermined time within 24 hours (1 day) to coincide 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 was collected after thorough stirring. The results are shown in Figures 1A to 20. The concentration of cell-secreted factors was analyzed using BioRad's Bioplex® or ELISA method. Measurements of each cell-secreted factor were performed according to the protocol specified for the assay kit used. When analyzing the supernatant concentration per unit cell count, the undifferentiation maintenance culture was completed, the culture supernatant cells were collected, and the cell number was counted. The concentration of each candidate protein in the supernatant per unit cell count was analyzed.

[0207] <Proteome Analysis> At the start of differentiation-inducing culture (day 0) and on days 3, 6, and 9 after the start of differentiation, a portion of the culture supernatant from each cell group was collected, and proteins in the obtained culture supernatant were concentrated using a centrifugal ultrafiltration filter (Amicon (registered trademark) Ultra 3K Device, manufactured by Merck Millipore). The concentrated solution was collected and passed through an ion exchange spin column (ProteoSpin) capable of removing specific proteins (albumin, α-antitrypsin, transferrin, and haptoglobulin). TM The concentrate was then subjected to an Abundant Serum Protein Depletion Kit (Norgen Biotek). Proteins in the concentrate were then digested with trypsin. Finally, the abundance of each protein was determined using liquid chromatography-mass spectrometry (LC-MS / MS analysis).

[0208] <Single Cell Gene Expression Analysis> Cells were collected immediately after the start of differentiation induction culture (i.e., immediately after the start of differentiation) 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. Next, the nucleotide sequence data of the single cell gene expression library was obtained using a next-generation sequencer Novaseq6000 (Illumina). The obtained nucleotide sequence data was processed using Cell Ranger software (10X Genomics) and converted into expression level data for each cell and each gene. Furthermore, the expression levels were normalized using Partek Flow software (Partek), and the expression counts of each gene per million counts of transcripts (CPM, or Counts Per Million) were calculated for each cell. The experimental results for each of the cell-secreted factors 1 to 25 are described in detail below.

[0209] <Cell secretory factor 1: Gas6 (Figures 1A, 1B, and 1C)> Using a research iPS cell line (201B7), changes in Gas6 during the differentiation induction process were measured over time for cell groups x1 and x2, which were cultured in a medium with different medium components during undifferentiation maintenance culture. Also, using a research iPS cell line (1231A3), changes in Gas6 during the differentiation induction process were measured over time for cell groups w1 and w2, which were cultured in a medium with different medium components during undifferentiation maintenance culture. (Experimental Results) The experimental results of culture experiment 1 using the 201B7 line are shown in Figure 1A. In cell group x1, which had a high differentiation induction efficiency, the Gas6 concentration was highest on day 1 of differentiation induction culture, and was higher than that of cell group x2, which had a low differentiation induction efficiency. From 0 to 216 hours after differentiation induction, Gas6 concentrations in the culture medium ranged from 27.1 pg / ml to 266.4 pg / ml (Figure 1A). The results of culture experiment 5 using the 1231A3 strain are shown in Figure 1B. In cell group w1, which showed high differentiation induction efficiency, the Gas6 concentration reached its highest level on day 1 of differentiation induction culture, and was higher than that of cell group w2, which showed low differentiation induction efficiency. In cell group w1, which showed high differentiation induction efficiency, the Gas6 concentration in the culture medium ranged from 115.5 pg / ml to 191.7 pg / ml from 24 to 72 hours after differentiation induction (Figure 1B). In addition, multiple experiments were performed using 201B7 under the experimental conditions of culture experiment 1, and the relationship between differentiation induction efficiency and Gas6 concentration in the culture supernatant on day 1 of differentiation induction was analyzed. The correlation coefficient was found to be greater than 0.7 (Figure 1C). These results suggest that the concentration of Gas6 in the culture medium can be used as an indicator of the differentiation efficiency of pluripotent stem cells into neural cells in the midbrain floor plate region.

[0210] <Cell-Secreted Factor 2: SerpinF1 (Figures 2A, 2B, and 2C)> Using a research iPS cell line (201B7), changes in SerpinF1 during the differentiation induction process were measured over time for cell groups x1 and x2, which were cultured in medium with different components during undifferentiation maintenance culture. Furthermore, using a research iPS cell line (1231A3), changes in SerpinF1 during the differentiation induction process were measured over time for cell groups w1 and w2, which were cultured in medium with different components during undifferentiation maintenance culture. (Experimental Results) The experimental results of culture experiment 1 using the 201B7 line are shown in Figure 2A. In cell group x1, which had a high differentiation induction efficiency, the SerpinF1 concentration was highest from days 9 to 12 of differentiation induction culture, and was higher than that of cell group x2, which had a low differentiation induction efficiency. From 0 to 288 hours after differentiation induction, the SerpinF1 concentration in the culture medium was confirmed to range from 1219.4 pg / ml to 52872.0 pg / ml (Figure 2A). The results of culture experiment 5 using the 1231A3 strain are also shown in Figure 2B. In cell group w1, which had a high differentiation induction efficiency, the SerpinF1 concentration reached its maximum from days 9 to 13 of differentiation induction culture, and was higher than that of cell group w2, which had a low differentiation induction efficiency. In cell group w1, which had a high differentiation induction efficiency, the SerpinF1 concentration in the culture medium was confirmed to range from 7961.0 pg / ml to 52658.0 pg / ml from 120 to 312 hours after differentiation induction (Figure 2B). In addition, multiple culture experiments were performed using the 201B7 strain under the experimental conditions of Culture Experiment 1, and the relationship between differentiation induction efficiency and the concentration in the supernatant on day 8 of culture was analyzed. The correlation coefficient between the SerpinF1 concentration and differentiation induction efficiency was found to be 0.7 or higher (Figure 2C). These results suggest that the SerpinF1 concentration in the culture medium can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0211] <Cell secretory factor 3: TFF3 (Figures 3A, 3B, and 3C)> Using a research iPS cell line (201B7), changes in TFF3 during the differentiation induction process were measured over time for cell groups x1 and x2, which were cultured in medium with different components during undifferentiation maintenance culture. Also, using a research iPS cell line (1231A3), changes in TFF3 during the differentiation induction process were measured over time for cell groups w1 and w2, which were cultured in medium with different components during undifferentiation maintenance culture. (Experimental Results) The experimental results of culture experiment 1 using the 201B7 line are shown in Figure 3A. In cell group x1, which had a high differentiation induction efficiency, the TFF3 concentration was highest on day 12 of differentiation induction culture, and was higher than that of cell group x2, which had a low differentiation induction efficiency. From 120 to 288 hours after differentiation induction, TFF3 concentrations in the culture medium ranged from 83.08 pg / ml to 1142.1 pg / ml (Figure 3A). The results of culture experiment 5 using the 1231A3 cell line are shown in Figure 3B. In cell group w1, which showed a high differentiation induction efficiency, the TFF3 concentration reached its highest level on day 13 of differentiation induction culture, and was higher than that of cell group w2, which showed a low differentiation induction efficiency. In cell group w1, which showed a high differentiation induction efficiency, the TFF3 concentration in the culture medium ranged from 47.3 pg / ml to 1025.0 pg / ml from 72 to 312 hours after differentiation induction (Figure 3B). Additionally, multiple culture experiments were performed using the 201B7 cell line under the experimental conditions of culture experiment 1. Analysis of the relationship between differentiation induction efficiency and supernatant TFF3 concentration after 11 days of culture revealed a correlation coefficient of 0.7 or higher (Figure 3C). These results suggest that the concentration of TFF3 in the culture medium can be used as an indicator of the differentiation efficiency of pluripotent stem cells into neural cells in the midbrain floor plate region.

[0212] <Cell-Secreted Factor 4: Cripto (Figure 4)> Using a research iPS cell line (201B7), changes in Cripto during the differentiation induction process were measured over time for cell groups x1 and x2, which were cultured in different medium components during undifferentiated maintenance culture. (Experimental Results) The experimental results of culture experiment 1 using the 201B7 line are shown in Figure 4. In cell group x1, which had a high differentiation induction efficiency, the Cripto concentration was highest on day 1 of differentiation induction culture, and was higher than that of cell group x2, which had a low differentiation induction efficiency. From 96 hours before differentiation induction (Day-4) to 72 hours after differentiation induction (Day 3), the Cripto concentration in the culture medium was confirmed to range from 118.0 pg / ml to 3153.7 pg / ml (Figure 4). In other experiments, in cell groups with high differentiation induction efficiency, the Cripto concentration in the culture medium was confirmed to range from 162.1 pg / ml to 1793.2 pg / ml between 24 and 72 hours after differentiation induction (not shown). These results suggest that the Cripto concentration in the culture medium from 96 hours before differentiation induction (Day 4) to 72 hours after differentiation induction (Day 3) can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0213] <Cell-Secreted Factor 5: WFDC2 (Figures 5A and 5B)> (A) Using a research iPS cell line (201B7), changes in WFDC2 during the differentiation induction process were measured over time for cell groups x1 and x2, which were cultured in different medium components during undifferentiated maintenance culture. (Experimental Results) Figure 5A shows the time course of WFDC2 concentration in the culture supernatant from culture experiment 1 using the 201B7 line. In cell group x1, which had a high differentiation induction efficiency, the WFDC2 concentration reached its maximum on day 2 of differentiation induction culture, and was higher than that of cell group x2, which had a low differentiation induction efficiency. From 48 hours before differentiation induction (Day-2) to 288 hours after differentiation induction (Day 12), the WFDC2 concentration in the culture medium was confirmed to range from 583.0 pg / ml to 26866.6 pg / ml (Figure 5A). In other experiments, in cell groups with high differentiation induction efficiency, WFDC2 concentrations in the culture medium were confirmed to range from 750.7 pg / ml to 14,167.8 pg / ml from 24 hours to 312 hours after differentiation induction (not shown). These results suggest that the WFDC2 concentration in the culture medium from 48 hours before differentiation induction (Day 2) to 312 hours after differentiation induction (Day 13) can be used as an indicator of the differentiation efficiency of pluripotent stem cells into neural cells in the midbrain floor plate region.

[0214] (B) The concentration of WFDC2 in the culture supernatant was measured in culture experiment 4 using a research iPS cell line (S line). (Experimental Results) Figure 5B shows a plot of the WFDC2 concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 for multiple culture supernatants from culture experiment 4 using the S line. Analysis of the data obtained for multiple supernatants revealed that the correlation coefficient between the WFDC2 concentration at the start of differentiation induction and the differentiation induction efficiency was 0.7 or higher (Figure 5B). These results suggest that the WFDC2 concentration in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0215] <Cell-Secreted Factor 6: CD30 (Figures 6A and 6B)> (A) Using a research iPS cell line (201B7), changes in CD30 during the differentiation induction process were measured over time for cell groups x1 and x2, which were cultured in different medium components during undifferentiated maintenance culture. (Experimental Results) Figure 6A shows the time course of CD30 concentration in the culture supernatant from culture experiment 1 using the 201B7 line. In cell group x1, which had a high differentiation induction efficiency, the CD30 concentration was highest on day 1 of differentiation induction culture, and was higher than that of cell group x2, which had a low differentiation induction efficiency. From 48 hours before differentiation induction (Day 2) to 48 hours after differentiation induction (Day 2), the CD30 concentration in the culture medium was confirmed to range from 5.1 pg / ml to 28.1 pg / ml (Figure 6A). In other experiments, in cell groups with high differentiation induction efficiency, CD30 concentrations in the culture medium were confirmed to range from 5.2 pg / ml to 27.0 pg / ml between 24 and 72 hours after differentiation induction (not shown). These results suggest that the CD30 concentrations in the culture medium from 48 hours before differentiation induction (Day 2) to 48 hours after differentiation induction (Day 2) can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0216] (B) The concentration of CD30 in the culture supernatant was measured in culture experiment 4 using a research iPS cell line (S line). (Experimental Results) Figure 6B shows a plot of the CD30 concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 for multiple culture supernatants from culture experiment 4 using the S line. Analysis of the data obtained for multiple supernatants revealed that the correlation coefficient between the CD30 concentration at the start of differentiation induction and the differentiation induction efficiency was 0.7 or higher. These results suggest that the CD30 concentration in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0217] <Cell-Secreted Factor 7: B2M (Figures 7A and 7B)> (A) Using a research iPS cell line (201B7), changes in B2M during the differentiation induction process were measured over time for cell groups x1 and x2, which were cultured in different medium components during undifferentiated maintenance culture. (Experimental Results) Figure 7A shows the time course of B2M concentration in the culture supernatant of culture experiment 1 using the 201B7 line. In cell group x1, which had a high differentiation induction efficiency, the B2M concentration at the start of differentiation induction was higher than the B2M concentration in cell group x2, which had a low differentiation induction efficiency, and the difference was the largest. From 48 hours before differentiation induction (Day-2) to 288 hours after differentiation induction (Day 12), the B2M concentration in the culture medium was confirmed to range from 656.4 pg / ml to 11,440.6 pg / ml (Figure 7A). In other experiments, in cell groups with high differentiation induction efficiency, B2M concentrations in the culture medium were confirmed to range from 3597.0 pg / ml to 9932.8 pg / ml between 24 and 312 hours after differentiation induction (not shown). These results suggest that the B2M concentrations in the culture medium from 48 hours before differentiation induction (Day 2) to 288 hours after differentiation induction (Day 12) can be used as an indicator of the differentiation efficiency of pluripotent stem cells into neural cells in the midbrain floor plate region.

[0218] (B) The concentration of B2M in the culture supernatant was measured in culture experiment 4 using a research iPS cell line (S line). (Experimental Results) Figure 7B shows a plot of the B2M concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 for multiple culture supernatants from culture experiment 4 using the S line. Analysis of the data obtained for multiple supernatants revealed that the correlation coefficient between the B2M concentration at the start of differentiation induction and the differentiation induction efficiency was 0.7 or higher. These results suggest that the B2M concentration in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0219] <Cell-Secreted Factor 8: CXCL6 (Figure 8)> The concentration of CXCL6 in the culture supernatant was measured in Culture Experiment 4 using a research iPS cell line (S line). (Experimental Results) Figure 8 shows a plot of the CXCL6 concentration in the culture supernatant on Day 0 of differentiation induction and the differentiation induction efficiency on Day 13 of differentiation induction for multiple culture supernatants from Culture Experiment 4 using the S line. Analysis of the data obtained for multiple supernatants revealed that the correlation coefficient between the CXCL6 concentration per unit cell number at the start of differentiation induction and the differentiation induction efficiency was 0.7 or higher. These results suggest that the CXCL6 concentration in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0220] <Cell-Secreted Factor 9: IGFBP6 (Figure 9)> The concentration of IGFBP6 in the culture supernatant was measured in culture experiment 4 using a research iPS cell line (S line). (Experimental Results) Figure 9 shows a plot of the IGFBP6 concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 for multiple culture supernatants from culture experiment 4 using the S line. Analysis of the data obtained for multiple supernatants revealed that the correlation coefficient between the IGFBP6 concentration per unit cell number at the start of differentiation induction and the differentiation induction efficiency was 0.7 or higher. This result suggests that the IGFBP6 concentration in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0221] <Cell-Secreted Factor 10: HGFR (Figure 10)> The concentration of HGFR in the culture supernatant was measured in culture experiment 4 using a research iPS cell line (S line). (Experimental Results) Figure 10 shows a plot of the HGFR concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 for multiple culture supernatants from culture experiment 4 using the S line. Analysis of the data obtained for multiple supernatants revealed that the correlation coefficient between the HGFR concentration per unit cell number at the start of differentiation induction and the differentiation induction efficiency was 0.7 or higher. This result suggests that the HGFR concentration in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0222] <Cell-Secreted Factor 11: SPARC (FIG. 11)> The concentration of SPARC in the culture supernatant was measured in Culture Experiment 4 using a research iPS cell line (S line). (Experimental Results) Figure 11 shows a plot of the SPARC concentration in the culture supernatant on Day 0 of differentiation induction and the differentiation induction efficiency on Day 13 of differentiation induction for multiple culture supernatants in Culture Experiment 4 using the S line. Analysis of the data obtained for multiple supernatants revealed that the correlation coefficient between the SPARC concentration per unit cell number at the start of differentiation induction and the differentiation induction efficiency was 0.7 or higher (FIG. 11). This result suggests that the SPARC concentration in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0223] <Cell-secreted factor 12: TRANCE (Figure 12)> The concentration of TRANCE in the culture supernatant was measured in culture experiment 4 using a research iPS cell line (S line). (Experimental Results) Figure 12 shows a plot of the TRANCE concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 for multiple culture supernatants from culture experiment 4 using the S line. Analysis of data obtained from multiple experiments showed that the correlation coefficient between the TRANCE concentration per unit cell number at the start of differentiation induction and the differentiation induction efficiency was 0.7 or higher. This result suggests that the TRANCE concentration in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0224] <Cell-Secreted Factor 13: PDGF-AA (Figure 13)> The concentration of PDGF-AA in the culture supernatant was measured in Culture Experiment 4 using a research iPS cell line (S line). (Experimental Results) Figure 13 shows a plot of the PDGF-AA concentration in the culture supernatant on Day 0 of differentiation induction and the differentiation induction efficiency on Day 13 of differentiation induction for multiple culture supernatants from Culture Experiment 4 using the S line. Analysis of data obtained from multiple experiments revealed that the correlation coefficient between the PDGF-AA concentration per unit cell number at the start of differentiation induction and the differentiation induction efficiency was 0.7 or higher. These results suggest that the concentration of PDGF-AA in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0225] <Cell-Secreted Factor 14: FGF13 (Figure 14)> The concentration of FGF13 in the culture supernatant was measured in culture experiment 4 using a research iPS cell line (S line). (Experimental Results) Figure 14 shows a plot of the FGF13 concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 for multiple culture supernatants from culture experiment 4 using the S line. Analysis of data obtained from multiple experiments revealed that the correlation coefficient between the FGF13 concentration per unit cell number at the start of differentiation induction and the differentiation induction efficiency was 0.7 or higher. These results suggest that the FGF13 concentration in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0226] <Cell-Secreted Factor 15: Kallikrein 6 (FIG. 15)> The concentration of Kallikrein 6 in the culture supernatant was measured in culture experiment 4 using a research iPS cell line (S line). (Experimental Results) Figure 15 shows a plot of the Kallikrein 6 concentration in the culture supernatant on differentiation induction day 0 and the differentiation induction efficiency on differentiation induction day 13 for multiple culture supernatants from culture experiment 4 using the S line. Analysis of data obtained from multiple experiments revealed that the correlation coefficient between the Kallikrein 6 concentration per unit cell number at the start of differentiation induction and the differentiation induction efficiency was 0.7 or higher (FIG. 15). These results suggest that the Kallikrein 6 concentration in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0227] <Cell-Secreted Factor 16: MMP2 (Figure 16)> The concentration of MMP2 in the culture supernatant was measured in Culture Experiment 4 using a research iPS cell line (S line). (Experimental Results) Figure 16 shows a plot of the MMP2 concentration in the culture supernatant on Day 0 of differentiation induction and the differentiation induction efficiency on Day 13 of differentiation induction for multiple culture supernatants from Culture Experiment 4 using the S line. Analysis of data obtained from multiple experiments revealed that the correlation coefficient between the MMP2 concentration per unit cell number at the start of differentiation induction and the differentiation induction efficiency was 0.7 or higher. These results suggest that the MMP2 concentration in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0228] <Cell-Secreted Factor 17: RAC2 (Figures 17A, 17B, and 17C)> Using a research iPS cell line (201B7), changes in RAC2 were measured over time in cell groups y1 and z1, which had high differentiation induction efficiency, and cell groups y2 and z2, which had low differentiation induction efficiency. Specifically, proteome analysis, ELISA concentration measurement, and single-cell gene expression analysis were performed. (Experimental Results) Cell group y1 had a Corin-positive cell rate of 58.2% on day 12 of differentiation-inducing culture. Meanwhile, cell group y2 had a Corin-positive cell rate of 34.1% on day 12 of differentiation-inducing culture. Furthermore, cell group z1 had a Corin-positive cell rate of 66.5% on day 12 of differentiation-inducing culture. Meanwhile, cell group z2 had a Corin-positive cell rate of 56.9% on day 12 of differentiation-inducing culture.

[0229] The results of the proteome analysis are shown in Figure 17A. On day 3 after the start of differentiation, the abundance (peak area) of RAC2 was 4.5E+05 in cell group y1, which had a high differentiation induction efficiency, whereas it was 2.1E+05 in cell group y2, which had a low differentiation induction efficiency.

[0230] The results of the ELISA assay are shown in Figure 17B. The Human Ras-Related c3 Botulinum Toxin Substrate 2 (RAC2) ELISA Kit (Abbexa) was used. On day 1 after the start of differentiation, the RAC2 concentration was 3551 pg / mL in cell group z1, which had a high differentiation induction efficiency, and 2335 pg / mL in cell group z2, which had a low differentiation induction efficiency.

[0231] The results of single-cell gene expression analysis are shown in Figure 17C. Immediately after the start of differentiation (day 0), the proportion of RAC2-expressing cells was 26% in cell group y1, which had a high differentiation induction efficiency, while it was 13% in cell group y2, which had a low differentiation induction efficiency. These results revealed that from immediately after the start of differentiation through day 3, the proportion of RAC2-expressing cells and the concentration of RAC2 in the supernatant were higher under conditions with a high differentiation induction efficiency than under conditions with a low differentiation efficiency. Furthermore, it was suggested that the concentration of RAC2 in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency from pluripotent stem cells to neural cells in the midbrain floor plate region.

[0232] <Cell secretory factor 18: TKT (Figures 18A, 18B, and 18C)> Using a research iPS cell line (201B7), changes in TKT were measured over time in cell groups y1 and z1, which had high differentiation induction efficiency, and cell groups y2 and z2, which had low differentiation induction efficiency. Specifically, proteome analysis, ELISA concentration measurement, and single-cell gene expression analysis were performed. (Experimental Results) Cell group y1 had a Corin-positive cell rate of 58.2% on day 12 of differentiation-inducing culture. Meanwhile, cell group y2 had a Corin-positive cell rate of 34.1% on day 12 of differentiation-inducing culture. Furthermore, cell group z1 had a Corin-positive cell rate of 66.5% on day 12 of differentiation-inducing culture. Meanwhile, cell group z2 had a Corin-positive cell rate of 56.9% on day 12 of differentiation-inducing culture.

[0233] The results of the proteome analysis are shown in Figure 18A. On day 3 after the start of differentiation, the abundance (peak area) of TKT was 1.9E+07 in cell group y1, which had a high differentiation induction efficiency, whereas it was 8.2E+06 in cell group y2, which had a low differentiation induction efficiency.

[0234] The results of the ELISA assay are shown in Figure 18B. The Human Transketolase Simple Step ELISA kit (TKT) (Abcam) was used. On day 1 after the start of differentiation, the TKT concentration was 708,311 pg / mL in cell group z1, which had a high differentiation induction efficiency, and 537,608 pg / mL in cell group z2, which had a low differentiation induction efficiency.

[0235] The results of single-cell gene expression analysis are shown in Figure 18C. Immediately after the start of differentiation (day 0), the average expression level of TKT was 770 CPM in cell group y1, which had a high differentiation induction efficiency, while it was 507 CPM in cell group y2, which had a low differentiation induction efficiency. These results revealed that from immediately after the start of differentiation through day 3, the gene expression level and supernatant concentration of TKT were higher under conditions with high differentiation efficiency than under conditions with low differentiation efficiency. Furthermore, it was suggested that the concentration of TKT in the culture medium at the start of differentiation induction can be used as an indicator of the differentiation efficiency of pluripotent stem cells into neural cells in the midbrain floor plate region.

[0236] <Cell-secreted factor 19: NPTX1 (Figures 19A and 19B)> Using a research iPS cell line (201B7), changes in NPTX1 during the differentiation induction process were measured over time for cell groups y1 and y2, and cell groups z1 and z2, which were cultured using different medium components during undifferentiation maintenance culture. Single-cell gene expression analysis and ELISA analysis of the concentration in the culture supernatant were performed. (Experimental Results) The results of single-cell gene expression analysis are shown in Figure 19A. The cell groups from culture experiment 2 were analyzed. In cell group y1, which had a high differentiation induction efficiency, the proportion of NPTX1-positive cells on day 0 of differentiation induction culture was 39%, while in cell group y2, which had a low differentiation induction efficiency, it was 5.1%. Furthermore, the counts per million (CPM), which is an index of the average RNA expression level, was 128 for cell group y1, which had a high differentiation induction efficiency, while it was 5.5 for cell group y2, which had a low differentiation induction efficiency. Therefore, it was revealed that under conditions of high differentiation induction efficiency, the expression level and proportion of NPTX1-expressing cells at the start of differentiation were high.

[0237] The results of ELISA analysis of the NPTX1 concentration in the culture supernatant are shown in Figure 19B. The culture supernatants obtained in culture experiments 2 and 3 were measured. The measurement kit used was an ELISA kit (product code: SEA298Hu) manufactured by Cloud Clone Corp. In cell groups with high differentiation induction efficiency, the NPTX1 concentration in the culture supernatant rose to 0.34 ng / mL (cell group y1) to 0.25 ng / mL (cell group z1) one day after the start of differentiation, whereas in cell groups with low differentiation induction efficiency, the concentration remained at 0.14 ng / mL (cell group y2) to 0.12 ng / mL (cell group z2) one day after the start of differentiation. Thus, the gene expression and secretion profiles of NPTX1 differed during the early stages of dopaminergic neuronal differentiation under conditions with high and low differentiation induction efficiency, demonstrating that this could serve as a marker for predicting the efficiency of dopaminergic neuronal differentiation induction at an early stage of differentiation.

[0238] <Cell-Secreted Factor 20: NT-3 (Figure 20)> Using a research iPS cell line (201B7), changes in NT-3 concentration were measured over time in a cell group with low differentiation induction efficiency and a cell group with high differentiation induction efficiency. (Experimental Results) In cell group x2, the percentage of Corin-positive cells was 24% on day 12 of differentiation-inducing culture. On the other hand, in cell group x1, the percentage of Corin-positive cells on day 12 of differentiation-inducing culture was 83%. Regarding NT-3 concentration in cell group x1, it was not detected in the culture supernatant samples collected 1 and 5 days after the start of differentiation, but it specifically increased in samples collected on days 2, 3, and 4 (Figure 20). On the other hand, in cell group x2, NT-3 was not detected in the culture supernatant samples collected on any day.

[0239] Based on the results of the cell-secreted factors 1 to 20 described above, it is considered preferable to set the reference concentration in the first method at, for example, 10 pg / ml. Furthermore, it is considered preferable to set the reference rate of change in the second method at, 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 being carried out.

[0240] <Other cell-secreted factors 21-25: PCSK1, ENPP2, PARP1, DX39B, AMD (not shown)> Using a research iPS cell line (201B7), changes in PCSK1, ENPP2, PARP1, DX39B, and AMD were measured over time in cell groups y1 and y2 of culture experiment 2, or cell groups z1 and z2 of culture experiment 3. Specifically, concentration measurements were performed using ELISA, single-cell gene expression analysis, and proteome analysis. (Experimental Results) The concentration of PCSK1 in the supernatant was measured using ELISA. A Human Proprotein Convertase Subtilisin / Kexin Type 1 (PCSK1) ELISA Kit (MyBioSource) was used for the measurements. The PCSK1 concentration on day 1 after the start of differentiation was 571 pg / mL in cell group z1, which had a high differentiation induction efficiency, and 378 pg / mL in cell group z2, which had a low differentiation induction efficiency.

[0241] By single cell gene expression analysis, the ratio of PCSK1 and ENPP2 expressing cells was determined.At the start of differentiation (day 0), the ratio of PCSK1 expressing cells was 8.4% in cell group y1 with high differentiation induction efficiency, while it was 3.9% in cell group y2 with low differentiation induction efficiency.At day 12 after the start of differentiation, the ratio of ENPP2 expressing cells was 24.7% in cell group y1 with high differentiation induction efficiency, while it was 47.6% in cell group y2 with low differentiation induction efficiency.

[0242] The abundance (peak area) of ENPP2, PARP1, DX39B, and AMD was determined by proteome analysis. The abundance of ENPP2 was 1.8E+07 in cell group y1, which had a high differentiation induction efficiency, on day 12 after the start of differentiation, while it was 3.7E+07 in cell group y2, which had a low differentiation induction efficiency. The abundances of PARP1, DX39B, and AMD were 7.3E+05, 8.0E+05, and 2.2E+05, respectively, in cell group y1, which had a high differentiation induction efficiency, on day 0 after the start of differentiation, while they were 15.6E+05, 17.1E+05, and 5.7E+04, respectively, in cell group y2, which had a low differentiation induction efficiency. From these results, it is clear that under the condition of high differentiation efficiency, compared with the condition of low differentiation efficiency, immediately after the start of differentiation, the concentration of PCSK1 in the supernatant and gene expression level are high, and after the start of differentiation, the 12th day, the existence amount of ENPP2 in the supernatant and the proportion of expressing cells are low.In addition, it is clear that at the start of differentiation, under the condition of high differentiation efficiency, compared with the condition of low differentiation efficiency, the existence amount of PARP1 and DX39B in the supernatant is low, and the existence amount of AMD in the supernatant is high.Therefore, it is suggested that the concentrations of PCSK1, ENPP2, PARP1, DX39B and AMD in culture medium can be used as an indicator of the differentiation efficiency of pluripotent stem cells into the neural cells of midbrain floor plate region.

[0243] <Summary> The results of cell-secreted factors 1 to 20 demonstrate that by monitoring the concentrations of cell-secreted factors secreted into the culture supernatant by pluripotent stem cells, i.e., during the differentiation process of pluripotent stem cells into neural cells of the midbrain floor plate region, it is possible to non-invasively monitor the state 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. The cell-secreted factors are Gas6, SerpinF1, TFF3, WFDC2, Cripto, CD30, B2M, CXCL6, IGFBP6, HGFR, SPARC, TRANCE, PDGF-AA, FGF13, Kallikrein6, MMP2, RAC2, TKT, NPTX1, and NT-3. Furthermore, the results for cell-secreted factors 21 to 25 revealed that PCSK1, ENPP2, PARP1, DX39B, and AMD can also be used as cell-secreted factors.

[0244] 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.

[0245] 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 non-invasively monitoring the state of cells in a culture medium during the differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region, comprising the steps of: measuring the concentration of a cell-secreted factor 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 cells of the midbrain floor plate region; comparing the measured concentration of the cell-secreted factor with a standard concentration; and 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 cell-secreted factor is equal to or greater than the standard concentration, wherein the culture supernatant is a culture supernatant collected from the culture medium at any time within 336 hours after the initiation of culturing the pluripotent stem cells.

2. A method for non-invasively monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region, comprising the steps of: measuring the concentration of a cell-secreted factor in each of a first culture supernatant and a second culture supernatant in a culture medium obtained by culturing the pluripotent stem cells in a medium containing a differentiation inducer into neural cells of the midbrain floor plate region; determining a rate of change in the concentration of the cell-secreted factor from the concentration of the cell-secreted factor in the first culture supernatant and the concentration of the cell-secreted factor in the second culture supernatant; comparing the absolute value of the rate of change in the concentration of the cell-secreted factor with a standard 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 concentration of the cell-secreted factor is equal to or greater than the standard rate of change, wherein the first culture supernatant is a culture supernatant collected from the culture medium at any time within 336 hours after the start of culturing the pluripotent stem cells, A method for non-invasively monitoring the state of cells in a culture medium during 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 at any time between 24 hours and 72 hours before collection of the first culture supernatant or at any time between 24 hours and 72 hours after collection of the first culture supernatant.

3. A method for non-invasively monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region, comprising the steps of: measuring the concentration of a cell-secreted factor in each of a first culture supernatant, a second culture supernatant, and a third culture supernatant in a 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; comparing the concentration of the cell-secreted factor 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 cells of the midbrain floor plate region when the concentration of the cell-secreted factor in the first culture supernatant is higher than the concentration of the cell-secreted factor in the second culture supernatant and the concentration of the cell-secreted factor in the third culture supernatant, A method for non-invasively monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region, wherein the first culture supernatant is a culture supernatant collected from the culture medium at any time within 336 hours after the initiation of culturing the pluripotent stem cells, the second culture supernatant is a culture supernatant collected from the culture medium at any time between 24 hours and 72 hours before collection of the first culture supernatant, and the third culture supernatant is a culture supernatant collected from the culture medium at any time between 24 hours and 72 hours after collection of the first culture supernatant.

4. A method for non-invasively monitoring the state of cells in a culture medium during 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 concentration of the cell-secreted factor is the amount of cell-secreted factor appearing in the culture medium per unit time.

5. A method for non-invasively monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells of the midbrain floor plate region as described in claim 1, wherein the reference concentration is the minimum concentration at which differentiation is successful.

6. A method for non-invasively monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region according to any one of claims 1 to 3, wherein the cell-secreted factors are any one or more selected from the group consisting of Gas6, SerpinF1, TFF3, WFDC2, Cripto, CD30, B2M, CXCL6, IGFBP6, HGFR, SPARC, TRANCE, PDGF-AA, FGF13, Kallikrein6, MMP2, RAC2, TKT, NPTX1, and NT-3.

7. A method for non-invasively monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells described in any one of claims 1 to 3 into neural cells of the midbrain floor plate region, wherein the pluripotent stem cells are iPS cells or ES cells.

8. A method for non-invasively monitoring the state of cells in a culture medium during 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 step of measuring the concentration of the cell-secreted factors includes multiple measurements of the concentration of one or more cell-secreted factors expected to appear within 336 hours after the start of culture.

9. A method for non-invasively monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells into neural cells in the midbrain floor plate region as described in claim 8, wherein the cell-secreted factors expected to appear within 336 hours after the start of the culture are any one or more selected from the group consisting of Gas6, SerpinF1, TFF3, WFDC2, Cripto, CD30, B2M, CXCL6, IGFBP6, HGFR, SPARC, TRANCE, PDGF-AA, FGF13, Kallikrein6, MMP2, RAC2, TKT, NPTX1, and NT-3.

10. A method for non-invasively monitoring the state of cells in a culture medium during differentiation of pluripotent stem cells described in any one of claims 1 to 3 into neural cells of the midbrain floor plate region, further comprising a step of determining whether or not the cells in the culture medium are abnormal.

11. 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 capable of inducing differentiation into neural cells of the midbrain floor plate region; (B) performing a method for non-invasively monitoring the state of cells in a culture medium during the differentiation of the pluripotent stem cells described in any one of claims 1 to 3 into neural cells of the midbrain floor plate region from the pluripotent stem cells during the period from 0 to 336 hours after the start of the culture of the pluripotent stem cells; and (C) deciding to continue to culture the cells in the culture medium 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 medium into neural cells of the midbrain floor plate region. (D) culturing neural cells in the midbrain floor plate region under culture conditions capable of inducing differentiation into the dopaminergic neural progenitor cells, thereby differentiating the cells into the dopaminergic neural progenitor cells or their precursor cells.

12. A method for producing dopaminergic neural progenitor cells or their precursor cells from the pluripotent stem cells described in claim 11, further comprising the step (E) of recovering neural cells in the midbrain floor plate region between steps (C) and (D).

13. A method for producing dopaminergic neural progenitor cells or their precursor cells from the pluripotent stem cells described in claim 11, wherein step (D) is carried out by selecting and recovering midbrain floor plate cells differentiated from neural cells in the midbrain floor plate region, and culturing the recovered midbrain floor plate cells under culture conditions capable of inducing differentiation into the dopaminergic neural progenitor cells.

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