JP2019154272A - Intracellular calcium dynamic evaluation system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an assay system capable of observing stable calcium dynamics in nerve cells and also enabling evaluation of changes in calcium dynamics during development. SOLUTION: Intracellular calcium of a nerve cell migrating from a neurosphere is measured, and the calcium dynamics of the nerve cell is evaluated. Typically, (i) a step of preparing a neurosphere in a culture vessel, (ii) a step of culturing under conditions for inducing differentiation into nerve cells, and (iii) loading migrating cells with a calcium indicator and then calcium. A step is performed to measure the signal intensity from the indicator and examine the changes in intracellular calcium. [Selection diagram] None

Description

  The present invention relates to a method for evaluating intracellular calcium kinetics and its use.

  Intracellular calcium plays an important role in the expression and control of various physiological functions. In nerve cells, it is important for the release of neurotransmitters and synaptic plasticity, and is also involved in the developmental stage (differentiation and maturation). For the analysis of intracellular calcium, calcium imaging using a fluorescent calcium reagent (calcium probe) (see, for example, Non-Patent Documents 1 and 2) is used. Hartfield et al. (Non-patent Document 3) perform calcium imaging using dopamine neurons differentiated from human PS cells. From about 4 weeks after induction of differentiation into dopamine neurons (6 weeks after induction of differentiation into nerves), clear intracellular calcium fluctuations are observed. In addition, it has been reported that calcium responses are observed by stimulating inositol triphosphate (IP3) receptors in early cultured neurons, but molecular pathways and development that contribute to spontaneous calcium fluctuations have been reported. Changes in the process have not been considered.

G. Grynkiewicz, M. Poenie, R. Y. Tsien, J. Biol. Chem., 1985, 260, 3440. K. R. Gee, K. A. Brown, W-N. U. Chen, J. Bishop-Stewart, D. Dray, I. Johnson, Cell Calcium, 2000, 27, 97 Elizabeth M. Hartfield et al., PLoS One. 2014 Feb 21; 9 (2): e87388.doi: 10.1371 / journal.pone.0087388.eCollection 2014.

  Conventionally, when calcium imaging is performed using neurons differentiated from human iPS cells, cell aggregates (embryoid bodies, neurospheres) are formed from human iPS cells, and then the cells are enzymatically and physically Nerve cells obtained by separating and culturing under differentiation-inducing conditions (dispersion culture) have been used (for example, Non-Patent Document 3 listed above). However, it is difficult to observe stable calcium dynamics due to the damage to the cells when they are subjected to dispersion culture, or because various cells with different degrees of differentiation / maturity coexist (cell heterogeneity). there were. In addition, it is common to measure intracellular calcium fluctuations and evaluate molecular pathways at a specific time (for example, mature neurons that have formed a neural network), and changes in intracellular calcium dynamics during development are examined. It has not been. Therefore, an object of the present invention is to provide an assay system that enables observation of stable calcium dynamics in nerve cells and also enables evaluation of changes in calcium dynamics during development and the use thereof.

  In order to solve the above problems, the present inventors have repeatedly studied and constructed a new measurement system. That is, instead of dispersion culture, neurospheres formed from pluripotent stem cells were cultured as they were to promote cell migration, and then the fluctuation of intracellular calcium was measured for the migrated cells. It can be said that culturing neurospheres and migrating cells reproduces or mimics the state in which differentiated neurons move in the living brain. In the case of this culture, it was expected that if the cells were identified by the distance from the neurosphere, a highly homogeneous cell population could be identified and stable intracellular calcium dynamics could be measured.

  As a result of verifying the effectiveness of the above measurement system, it was possible to observe fluctuations in intracellular calcium with stability and accuracy exceeding expectations by short-time measurement. This result confirms that the measurement system is extremely effective as a means for observing and evaluating calcium dynamics in nerve cells. In addition, it should be noted that according to the above measurement system, it is also possible to capture a phenomenon in which a molecular pathway contributing to calcium fluctuation is changed (that is, a change in the generation process) as in the living brain. This fact is effective in grasping and evaluating calcium dynamics in neurons and their changes at the developmental stage (neural circuit formation process), and is useful for basic research or development of new drugs. It shows that. For example, when neurospheres are induced to differentiate into dopamine neurons, spontaneous calcium dynamics can be evaluated via specific pathways (IP3 receptor pathway or voltage-dependent calcium channel (VDCC) pathway) selectively. Allows screening of new drugs targeting the pathway.

The following invention is based on the above results and considerations.
[1] A method for evaluating calcium dynamics in nerve cells, comprising measuring intracellular calcium in nerve cells migrated from neurospheres.
[2] The calcium kinetics evaluation method according to [1], comprising the following steps (i) to (iii):
(I) preparing a neurosphere in a culture vessel;
(Ii) culturing under conditions for inducing differentiation into nerve cells,
(Iii) A step of measuring the intracellular calcium fluctuation by measuring the signal intensity from the calcium indicator after loading the migrated cells with the calcium indicator.
[3] The method for evaluating calcium kinetics according to [1] or [2], wherein the neurosphere is derived from a pluripotent stem cell.
[4] The method for evaluating calcium dynamics according to [3], wherein the pluripotent stem cell is an induced pluripotent stem cell.
[5] The calcium kinetics evaluation method according to any one of [1] to [4], wherein the cells constituting the neurosphere are non-normal cells.
[6] The calcium kinetics evaluation method according to [5], wherein the non-normal cell is a disease cell having a genetic characteristic of the target disease.
[7] The method for evaluating calcium dynamics according to [6], wherein the diseased cell is a patient-derived cell or a cell created by genetic manipulation.
[8] The calcium kinetics evaluation method according to any one of [1] to [7], wherein the nerve cells are dopamine neurons.
[9] The method for evaluating calcium dynamics according to any one of [1] to [8], wherein intracellular calcium is measured over time.
[10] The calcium kinetics evaluation method according to any one of [1] to [9], wherein the neurosphere is prepared by a method including the following steps (1) and (2):
(1) culturing pluripotent stem cells in the presence of a TGF-β family inhibitor, a GSK3β inhibitor and a BMP inhibitor;
(2) A step of subjecting the cells obtained in step (1) to suspension culture in the presence of a TGF-β family inhibitor, GSK3β inhibitor, FGF8 and hedgehog signal agonist and normal oxygen partial pressure to form neurospheres .
[11] In any one of [2] to [10], in step (iii), intracellular calcium is measured for a plurality of cells having the same migration distance, and the measurement results are totaled to evaluate the variation in intracellular calcium. The method for evaluating calcium dynamics according to one item.
[12] In step (iii), occurrence frequency or frequency of intracellular calcium concentration increase event, maximum rate of change, or duration, time constant or half-life in increasing / decreasing intracellular calcium concentration, and increasing intracellular calcium concentration The method for evaluating calcium kinetics according to any one of [2] to [10], wherein fluctuations in intracellular calcium are examined based on one or more items selected from the group consisting of an integral value of a signal change rate in an event. .
[13] In any one of [2] to [10], in step (iii), after adding a substance that acts directly or indirectly on a pathway involved in intracellular calcium dynamics, the signal intensity from the calcium indicator is measured The method for evaluating calcium kinetics according to claim 1.
[14] A drug evaluation method comprising the following steps (I) to (III):
(I) a step of preparing a neurosphere in a culture vessel;
(II) culturing under conditions for inducing differentiation into nerve cells,
(III) loading the migrated cells with a calcium indicator;
(IV) adding a test substance;
(V) A step of measuring the signal intensity from the calcium indicator, examining changes in intracellular calcium, and evaluating the efficacy or toxicity of the test substance.
[15] In step (V), the signal intensity is measured before and after the addition of the test substance, the measurement result before the test substance is added and the measurement result after the test substance is added, and the efficacy or toxicity of the test substance is evaluated. The drug evaluation method according to [14].
[16] In step (V), the measurement result of the signal intensity is compared with the measurement result of the signal intensity of the cells treated under the same conditions except that the test substance is not added, and the efficacy or toxicity of the test substance is evaluated. The drug evaluation method according to [14].
[17] The drug according to any one of [14] to [16], wherein a substance that directly or indirectly acts on a pathway involved in intracellular calcium dynamics is added before the test substance is added. Evaluation methods.
[18] The substance is a promoter or inhibitor of inositol triphosphate (IP3) -dependent calcium mobilization, or a promoter or inhibitor of L-type voltage-dependent calcium channel (VDCC) -dependent calcium influx. ] The medicine evaluation method of description.
[19] The drug evaluation method according to [18], wherein the nerve cell is a dopamine neuron, and the signal intensity in step (V) is measured between the start of culture under differentiation-inducing conditions and the seventh day.
[20] The neuron is a dopamine neuron, and the signal intensity of step (V) is measured from day 14 to day 30 after the start of culture under differentiation-inducing conditions. Drug evaluation method.

Results of intracellular calcium imaging. Addition of 2-APB (8 days after initiation of differentiation induction) suppressed intracellular calcium fluctuations in iPS cell-derived dopaminergic neurons (right). The left image is before 2-APB addition. By photographing for 5 minutes using the calcium indicator Fluo4-AM. Scanning: 0.5 fps (flame per sec), image compression: 50 fps. Results of intracellular calcium imaging. The addition of 2-APB (14 days after the start of differentiation induction) slightly affected intracellular calcium fluctuations in iPS cell-derived dopaminergic neurons. By photographing for 30 minutes using the calcium indicator Fluo4-AM. Scanning: 0.5 fps (flame per sec), image compression: 50 fps. Results of intracellular calcium imaging. Addition of TTX (14 days after initiation of differentiation induction) suppressed intracellular calcium fluctuations in iPS cell-derived dopaminergic neurons. By photographing for 30 minutes using the calcium indicator Fluo4-AM. Scanning: 1 fps (flame per sec), image compression: 100 fps. Results of intracellular calcium imaging. Nimodipine addition (15 days after the start of differentiation induction) suppressed intracellular calcium fluctuations in iPS cell-derived dopaminergic neurons (left). In contrast, addition of FPL 64176 (day 17 after the start of differentiation induction) increased intracellular calcium fluctuations in iPS cell-derived dopaminergic neurons (right). By taking 30 minutes (left) or 15 minutes (right) with the calcium indicator Fluo4-AM. Scanning: 1 fps (flame per sec), image compression: 100 fps. Summary of effects of each drug on calcium dynamics. TTX (sodium channel inhibitor, Wako), 2-APB (cell permeable allosteric inhibitor for IP3-induced Ca ²⁺ release, Sigma), Nimodipine (L-type VDCC inhibitor), FPL 64176 (putative L-type VDCC activator , Tocris). 100 μM 2-APB strongly induces an increase in intracellular Ca ²⁺ levels and causes cell contraction. Matrigel coat conditions are as follows. 100 / w: Matrigel 1: 100, 1 wash (PBS). 30 / w: Matrigel 1:30, 1 wash (PBS). 30 / nw: Matrigel 1:30, not washed. A method for quantitative analysis of intracellular calcium imaging. Analysis of calcium dynamics based on calcium events. Details of waveform analysis (example).

1. Calcium kinetic evaluation method The first aspect of the present invention provides a method for evaluating calcium kinetics of nerve cells (hereinafter referred to as “calcium kinetic evaluation method of the present invention”). In the present invention, intracellular calcium of nerve cells migrated from neurospheres is measured. That is, instead of using cells collected from neurospheres or cells obtained by inducing differentiation thereof, cells that migrate from neurospheres are targeted for measurement.

  The neurosphere is a spherical neuronal cell mass (cell aggregate) including neural stem cells / undifferentiated neurons. The neurosphere used in the present invention can be prepared according to a method already reported. Various methods were developed to improve the neurosphere method reported by Weiss et al. (Science 255, 1707-1710, 1992), and as a means of selectively proliferating neural stem cells and neural progenitor cells, or in vitro, neurons and glia Widely used as a means for inducing differentiation of cells and the like (for example, Reynolds BA and Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 1992 Mar 27; 255 (5052): 1707 -10 .; Uemura T. et al., Transplantation of induced pluripotent stem cell-derived neurospheres for peripheral nerve repair.Biochem Biophys Res Commun. 2012 Mar 2; 419 (1): 130-5 .; Kawano E. et al. , Induction of neural crest cells from human dental pulp-derived induced pluripotent stem cells.Biomed Res. 2017; 38 (2): 135-147.doi: 10.2220 / biomedres.38.135 .; Kucia M. et al., Cells enriched in markers of neural tissue-committed stem cells reside in the bone marrow and are mobilized int o The peripheral blood following stroke. See Leukemia, 2006, 20: 18-28).

  When neurospheres are cultured under predetermined conditions (for example, conditions that promote differentiation into dopamine neurons), some cells begin to move away from the neurospheres. The cells that have migrated (moved) in this way are the objects of measurement.

Typically, in the calcium kinetics evaluation method of the present invention, the following steps (i) to (iii) are performed.
(I) Step of preparing neurospheres in a culture vessel (ii) Step of culturing under conditions for inducing differentiation into nerve cells (iii) After loading a calcium indicator on migrated cells, measure the signal intensity from the calcium indicator , Step to examine the fluctuation of intracellular calcium

First, the neurosphere is prepared in a culture vessel such as a dish, petri dish, tissue culture dish, multi-dish, microplate, microwell plate, multiplate, multiwell plate, petri dish (step (i)). Matrigel ^TM (BD), poly -D- lysine, poly -L- lysine, collagen, gelatin, laminin, heparan sulfate proteoglycans, entactin, or the coated treated culture vessel by a combination of two or more among these used, You may decide to improve the adhesiveness of the cell to a culture surface.

  Preferably, neurospheres derived from pluripotent stem cells are prepared and used for the calcium kinetic evaluation method of the present invention. “Derived from a pluripotent stem cell” is synonymous with being formed by inducing differentiation of a pluripotent stem cell. The term “pluripotent stem cells” refers to the ability to differentiate into all the cells that make up a living body (differentiation pluripotency) and the ability to generate daughter cells that have the same differentiation potential as self through cell division (self-replication ability) ). Pluripotency can be evaluated by transplanting cells to be evaluated into nude mice and testing for the presence or absence of teratoma containing each of the three germ layers (ectodermal, mesoderm, and endoderm). it can.

  Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells), which have both differentiation pluripotency and self-renewal ability. As long as it is a cell, it is not limited to this. Preferably, ES cells or iPS cells are used. More preferably iPS cells are used. The pluripotent stem cells are preferably mammalian cells (for example, primates such as humans and chimpanzees, rodents such as mice and rats), particularly preferably human cells. Therefore, in the most preferred embodiment of the present invention, human iPS cells are used as pluripotent stem cells.

  ES cells can be established, for example, by culturing an early embryo before implantation, an inner cell mass constituting the early embryo, a single blastomere, etc. (Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Thomson, JA et al., Science, 282, 1145-1147 (1998)). As an early embryo, an early embryo produced by nuclear transfer of a somatic cell nucleus may be used (Wilmut et al. (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280, 1256). (1998)), Akira Iriya et al. (Protein Nucleic Acid Enzyme, 44, 892 (1999)), Baguisi et al. (Nature Biotechnology, 17, 456 (1999)), Wakayama et al. (Nature, 394, 369 (1998)). Nature Genetics, 22, 127 (1999); Proc. Natl. Acad. Sci. USA, 96, 14984 (1999)), Rideout III et al. (Nature Genetics, 24, 109 (2000), Tachibana et al. Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013) in press) Parthenogenetic embryos may be used as the initial locus (Kim et al. (Science, 315, 482-486 (2007)) Nakajima et al. (Stem Cells, 25, 983-985 (2007)), Kim et al. (Cell Stem Cell, 1, 346-352 (2007)), Revazova et al. (Cloning Stem Cells, 9, 432- 449 (2007) ), Revazova et al. (Cloning Stem Cells, 10, 11-24 (2008)) In addition to the papers listed above, Strelchenko N., et al. Reprod Biomed Online. 9: 623-629 Klimanskaya I., et al. Nature 444: 481-485, 2006; Chung Y., et al. Cell Stem Cell 2: 113-117, 2008; Zhang X., et al Stem Cells 24: 2669-2676 , 2006; Wassarman, PM et al. Methods in Enzymology, Vol. 365, 2003, etc. It should be noted that fused ES cells obtained by cell fusion of ES cells and somatic cells are also included in embryonic stem cells.

  Some ES cells are available from preservation institutions or are commercially available. For example, human ES cells can be obtained from the Institute of Regenerative Medicine, Kyoto University (for example, KhES-1, KhES-2 and KhES-3), WiCell Research Institute, ESI BIO and the like.

  EG cells can be established by culturing primordial germ cells in the presence of LIF, bFGF, SCF, etc. (Matsui et al., Cell, 70, 841-847 (1992), Shamblott et al., Proc. Natl. Acad. Sci. USA, 95 (23), 13726-13731 (1998), Turnpenny et al., Stem Cells, 21 (5), 598-609, (2003)).

  “Artificial pluripotent stem cells (iPS cells)” are differentiated pluripotent cells created by reprogramming somatic cells (eg, fibroblasts, skin cells, lymphocytes, etc.) by introducing reprogramming factors. And self-replicating cells. iPS cells are similar to ES cells. Somatic cells used for the production of iPS cells are not particularly limited, and may be differentiated somatic cells or undifferentiated stem cells. iPS cells can be prepared by various methods reported so far. In addition, it is naturally assumed that an iPS cell production method developed in the future will be applied. Examples of cells that can be used for the production of iPS cells, that is, cells derived from iPS cells include lymphocytes (T cells, B cells), fibroblasts, epithelial cells, endothelial cells, mucosal epithelial cells, mesenchymal cells Examples include stem cells, hematopoietic stem cells, adipose stem cells, dental pulp stem cells, and neural stem cells.

  The most basic method for producing iPS cells is to introduce four factors, transcription factors Oct3 / 4, Sox2, Klf4 and c-Myc, into cells using viruses (Takahashi K, Yamanaka S : Cell 126 (4), 663-676, 2006; Takahashi, K, et al: Cell 131 (5), 861-72, 2007). Human iPS cells have been reported to be established by introducing four factors, Oct4, Sox2, Lin28 and Nonog (Yu J, et al: Science 318 (5858), 1917-1920, 2007). 3 factors except c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26 (1), 101-106, 2008), Oct3 / 4 and Klf4 (Kim JB, et al: Nature 454 (7204) , 646-650, 2008), or the establishment of iPS cells by introducing only Oct3 / 4 (Kim JB, et al: Cell 136 (3), 411-419, 2009) has been reported. In addition, a method for introducing a protein, which is an expression product of a gene, into a cell (Zhou H, Wu S, Joo JY, et al: Cell Stem Cell 4, 381-384, 2009; Kim D, Kim CH, Moon JI, et al : Cell Stem Cell 4, 472-476, 2009). On the other hand, by using the inhibitor BIX-01294 for histone methyltransferase G9a, the histone deacetylase inhibitor valproic acid (VPA) or BayK8644, production efficiency can be improved and factors to be introduced can be reduced. (Huangfu D, et al: Nat. Biotechnol. 26 (7), 795-797, 2008; Huangfu D, et al: Nat. Biotechnol. 26 (11), 1269-1275, 2008; Silva J , et al: PLoS. Biol. 6 (10), e 253, 2008). Studies on gene transfer methods are also underway, and in addition to retroviruses, lentiviruses (Yu J, et al: Science 318 (5858), 1917-1920, 2007), adenoviruses (Stadtfeld M, et al: Science 322 (5903 ), 945-949, 2008), plasmid (Okita K, et al: Science 322 (5903), 949-953, 2008), transposon vector (Woltjen K, Michael IP, Mohseni P, et al: Nature 458, 766- 770, 2009; Kaji K, Norrby K, Pac a A, et al: Nature 458, 771-775, 2009; Yusa K, Rad R, Takeda J, et al: Nat Methods 6, 363-369, 2009), or A technique using an episomal vector (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) has been developed.

  Cells that have undergone transformation (reprogramming) into iPS cells are expressed pluripotent stem cell markers (undifferentiation markers) such as Fbxo15, Nanog, Oct / 4, Fgf-4, Esg-1, and Cript Etc. can be selected as an index.

  iPS cells can also be provided from, for example, Kyoto University or the RIKEN BioResource Center.

  Pluripotent stem cells can be maintained in vitro by known methods. Maintaining pluripotent stem cells by serum-free culture using serum substitutes or feeder-free cell culture when it is desired to provide highly safe cells, such as when clinical application is considered It is preferable. If serum is used (or combined), autologous serum (ie recipient serum) may be used. Serum replacements can contain, for example, albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3 ′ thiol glycerol, or equivalents thereof. Serum replacement can be prepared by known methods (see, eg, WO 98/30679). A commercially available serum replacement can also be used. Examples of commercially available serum substitutes include KSR (Invitrogen), Chemically-defined Lipid concentrated (Gibco) and Glutamax (Gibco).

The cells constituting the neurosphere in step (i) are normal cells or non-normal cells. In other words, a neurosphere composed of normal cells or a neurosphere composed of non-normal cells is used. Normal cells are typically cells from healthy individuals. “Non-normal cells” are cells that are in contrast to normal cells, and are cells that are no longer in their original state due to genetic mutations or chromosomal abnormalities. An example of a non-normal cell is a cell having a genetic abnormality characteristic of a specific disease (hereinafter referred to as “target disease”), and a cell derived from a patient suffering from the target disease corresponds to a non-normal cell. Examples of patient-derived cells include differentiation induction of cells collected from patients or their passage cells, and patient-derived artificial pluripotent stem (iPS) cells (iPS cells created using cells collected from patients). The obtained differentiated cells. In addition to patient-derived cells, cells introduced with genetic abnormalities characteristic of the target disease (for example, by introducing gene mutations into undifferentiated cells such as iPS cells by genetic manipulation such as genome editing, etc. Obtained differentiated cells) and cells obtained by introducing gene mutations into differentiated cells can also be adopted as non-normal cells. Examples of target diseases are Timothy syndrome, autism spectrum disorder (ASD), schizophrenia, Parkinson's disease, anxiety disorder, bipolar disorder, and drug dependence. Examples of genetic abnormalities are voltage-dependent Ca. ²⁺ channel (VDCC) main subunit (CACNA1C, CACNA1D, etc.) gene or subunit (CACNA2D1, CACNA2D2, CACNA2D3, CACNA2D4, CACNB2, CACNB4, etc.).

In a preferred embodiment, as the neurosphere in step (i), a neurosphere prepared by a method including the following steps (1) and (2) is used. The neurosphere is suitable for obtaining dopamine neurons.
(1) A step of culturing pluripotent stem cells in the presence of a TGF-β family inhibitor, a GSK3β inhibitor and a BMP inhibitor (2) The cells obtained in step (1) are transformed into a TGF-β family inhibitor, GSK3β A step of forming a neurosphere by suspension culture in the presence of an inhibitor, FGF8 and a hedgehog signal agonist and under normal oxygen partial pressure.

Step (1)
In step (1), pluripotent stem cells are used. The “pluripotent stem cell” is as described above. In step (1), pluripotent stem cells are cultured in the presence of a TGF-β family inhibitor, a GSK3β inhibitor, and a BMP inhibitor. That is, pluripotent stem cells are cultured using a medium to which a TGF-β family inhibitor, a GSK3β inhibitor and a BMP inhibitor are added. Step (1) aims to enhance the neuronal differentiation ability of pluripotent stem cells.

  The medium can be prepared using a medium used for culturing mammalian cells as a basal medium. As the basal medium, for example, BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham medium, Ham's F-12 medium , RPMI1640 medium, Fischer's medium, Neurobasal medium, and mixed media thereof are not particularly limited as long as they can be used for culturing mammalian cells. In one embodiment, a mixed medium of IMDM medium and Ham's F-12 medium is used. The mixing ratio is a volume ratio, for example, IMDM: Ham's F-12 = O.8 to 1.2: 1.2 to 0.8.

  A TGF-β family inhibitor, a GSK3β inhibitor, and a BMP inhibitor are added to the medium. A TGF-β family inhibitor is a substance that inhibits TGF-β signaling through binding between TGF-β and a TGF-β receptor. TGF-β inhibitors include proteinaceous inhibitors and small molecule inhibitors. Examples of proteinaceous inhibitors are anti-TGF-β neutralizing antibodies and anti-TGF-β receptor neutralizing antibodies. Examples of small molecule inhibitors are SB431542 (4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] -benzamide or its Hydrate), SB202190 (4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories). Preferably, SB431542 is used. The concentration of the TGF-β family inhibitor (addition amount to the medium) is not particularly limited as long as the purpose of enhancing the neuronal differentiation ability of pluripotent stem cells is achieved. 0.5 μM to 20 μM, preferably 1 μM to 10 μM. The optimum concentration can be set through preliminary experiments. Rather than keeping the TGF-β family inhibitor concentration constant throughout the entire culture period, changes in the TGF-β family inhibitor concentration may be provided, for example, by increasing the TGF-β family inhibitor concentration stepwise.

  As a GSK3β inhibitor, CHIR99021 (6-[[2-[[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] ethyl]) Amino] nicotinonitrile), SB-415286 (3-[(3-chloro-4-hydroxyphenyl) amino] -4- (2-nitrophenyl) -1H-pyrrole-2,5-dione), SB-2167 , Indirubin-3′-Monoxime, Kenpaullone, BIO (6-bromoindirubin-3′-oxime) and the like can be used. Preferably, CHIR99021 is used. The concentration of GSK3β inhibitor (addition amount to the medium) is not particularly limited as long as the purpose of enhancing the neuronal differentiation ability of pluripotent stem cells is achieved, but when the concentration is shown by taking CHIR99021 as an example, for example, 0.5 μM to 20 μM, preferably 1 μM to 10 μM. The optimum concentration can be set through preliminary experiments. Instead of making the GSK3β inhibitor concentration constant throughout the entire culture period, the GSK3β inhibitor concentration may be changed, for example, by increasing the GSK3β inhibitor concentration stepwise.

  A BMP inhibitor is a substance that inhibits BMP signaling (BMP signaling) through binding between BMP (bone morphogenetic protein) and a BMP receptor (type I or type II). BMP inhibitors include proteinaceous inhibitors and small molecule inhibitors. Examples of proteinaceous inhibitors are the natural inhibitors Noggin, chordin, follistatin and the like. Examples of small molecule inhibitors include Dorsomorphin (6- [4- (2-piperidin-1-ylethoxy) phenyl] -3-pyridin-4-ylpyrazolo [1,5-a] pyrimidine) and its derivatives, LDN-193189 (4- (6- (4-piperazin-1-yl) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline) and its derivatives. These compounds are commercially available (for example, available from Sigma-Aldrich and Stemgent) and are readily available. Preferably, Dorsomorphin is used. The concentration of BMP inhibitor (addition amount to the medium) is not particularly limited as long as the purpose of enhancing the neuronal differentiation ability of pluripotent stem cells is achieved, but when the concentration is shown by taking Dorsomorphin as an example, for example, 0.5 μM to 20 μM, preferably 1 μM to 10 μM. The optimum concentration can be set through preliminary experiments. Instead of making the BMP inhibitor concentration constant throughout the entire culture period, changes in the BMP inhibitor concentration may be provided, for example, by increasing the BMP inhibitor concentration stepwise.

  You may add another component to a culture medium as needed. Examples of components that can be added include insulin, iron sources (such as transferrin), minerals (such as sodium selenate), sugars (such as glucose), organic acids (such as pyruvic acid, lactic acid, etc.), serum proteins (such as albumin) Etc.), amino acids (eg L-glutamine etc.), reducing agents (eg 2-mercaptoethanol etc.), vitamins (eg ascorbic acid, d-biotin etc.), antibiotics (eg streptomycin, penicillin, gentamicin etc.), buffering agents (For example, HEPES).

Pluripotent stem cells are usually subjected to adhesion culture. Adhesive culture is a culture that is in contrast to suspension culture, and is typically two-dimensional culture (planar culture) under adhesive conditions. However, Matrigel ^™ (BD) or the like may be used for three-dimensional culture. For adhesion culture, for example, a dish, petri dish, tissue culture dish, multi-dish, microplate, microwell plate, multiplate, multiwell plate, chamber slide, petri dish or the like can be used. Matrigel ^™ (BD), poly-D-lysine, poly-L-lysine, collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, or two of these to enhance cell adhesion to the culture surface An incubator coated by the above combination may be used.

  Pluripotent stem cells can be cultured in the presence or absence of feeder cells, but when it is desired to provide highly safe cells, such as when clinical application is in view Is preferably cultured in the absence of feeder cells (feeder cell culture). Examples of feeder cells are MEF (mouse fetal fibroblasts), STO cells (mouse fetal fibroblast cell line), and SNL cells (STO cell subclone).

Culture temperature, C0 ₂ concentration, other culture conditions such as 0 ₂ concentration can be set as appropriate. The culture temperature is, for example, about 30 to 40 ° C, preferably about 37 ° C. The CO ₂ concentration is for example about 1-10%, preferably about 5%. Moreover, what is necessary is just to culture | cultivate under normal oxygen partial pressure. Although it may vary depending on other conditions (humidity, CO ₂ concentration, etc.), the oxygen concentration in the case of “under normal oxygen partial pressure” is typically about 18% to about 22%. The details of “normal oxygen partial pressure” will be described later.

  The period (culture period) of step (1) is 4 days or more, specifically, for example, 4 days to 20 days, preferably 6 days to 14 days. If the culture period is too short, neurosphere formation ability is reduced.

  It may be subcultured as necessary. For example, cells are collected at the stage of subconfluence or confluence, a part of the cells are seeded in another incubator, and the culture is continued. A cell dissociation solution or the like may be used for cell recovery. As the cell dissociation solution, for example, proteolytic enzymes such as EDTA-trypsin, collagenase IV, metalloprotease and the like can be used alone or in appropriate combination. Those having low cytotoxicity are preferred. As such a cell dissociation solution, for example, commercially available products such as dispase (Adia), TrypLE (Invitrogen), or Accutase (MILLIPORE) are available. The recovered cells may be subjected to subculture after being treated with a cell strainer or the like so as to be in a dispersed (discrete) state.

  As a result of step (1), the neuronal differentiation ability of pluripotent stem cells is enhanced. Increased nerve differentiation ability can be confirmed by using as an indicator that expression of nervous system markers (Sox2, Nestin, Sox1, etc.) is increased as compared to before the start of step (1). Moreover, you may utilize the expression of an undifferentiation marker for evaluation that nerve differentiation ability was enhanced.

  Usually, the cells after step (1) are once collected and then proceed to the next culture (step (2)). The collection operation can be performed in the same manner as the collection operation during subculture.

Step (2)
In this step, the cells obtained in step (1) are suspended in the presence of TGF-β family inhibitor, GSK3β inhibitor, FGF8 and hedgehog signal agonist and under normal oxygen partial pressure to form neurospheres. . That is, the cells after step (1) are cultured using a medium to which a TGF-β family inhibitor, a GSK3β inhibitor, FGF8 and a hedgehog signal agonist are added and under a normal oxygen partial pressure. Step (2) aims to induce differentiation along the neural cell lineage. Items that are not specifically mentioned (usable basic medium, usable TGF-β family inhibitor, GSK3β inhibitor, other components that can be added to the medium, etc.) are the same as in step (1). Omitted.

  For suspension culture, for example, flask, tissue culture flask, dish, petri dish, tissue culture dish, multi-dish, microplate, microwell plate, micropore, multiplate, multiwell plate, chamber slide, petri dish, tube , Trays, culture bags, roller bottles and the like can be used. In order to enable culturing under non-adhesive conditions, it is preferable to use an incubator having a non-cell-adhesive culture surface. Applicable incubators include those whose surface (culture surface) is treated so as to be non-cell-adhesive, and treatment for improving cell adhesion (for example, coating treatment with an extracellular matrix, etc.) Those not applied to the culture surface) can be mentioned. In suspension culture, it is only necessary to maintain a non-adhered state of the cells with respect to the incubator, and static culture may be employed, or swirl culture or shaking culture may be employed.

  Among the components added to the medium, the TGF-β family inhibitor and the GSK3β inhibitor are as described above. Also in this step, it is preferable to use SB431542 as a TGF-β family inhibitor and CHIR99021 as a GSK3β inhibitor. The concentration in the culture medium when SB431542 is used is, for example, 0.5 μM to 20 μM, preferably 1 μM to 10 μM. Similarly, the concentration in the medium when CHIR99021 is used is, for example, 0.5 μM to 20 μM, preferably 1 μM to 10 μM.

  FGF8 is a member of the fibroblast growth factor family. FGF8 is involved in the control of vertebrate brain formation and is required for localization to the midbrain. As long as the object of the present invention can be achieved, FGF8 derived from various mammals can be used. However, it is preferable to match the origin (animal species) with the pluripotent stem cells to be used. Therefore, human FGF8 is preferably used when human pluripotent stem cells are used. Human FGF8 means that the human has the amino acid sequence of FGF8 that is naturally expressed in vivo, and may be a recombinant. As a representative amino acid sequence of human FGF8, NP_006110.1 (fibroblast growth factor 8 isoform B precursor [Homo sapiens].) Can be exemplified by the NCBI accession number. The concentration of FGF8 (addition amount to the medium) is not particularly limited as long as the purpose of inducing differentiation along the neural cell lineage is achieved, but for example, 1 ng / ml to 5 μg / ml, preferably 10 to 500 ng / ml More preferably, it is 50 to 400 ng / ml. The optimum concentration can be set through preliminary experiments.

  The hedgehog signal agonist is not particularly limited as long as it promotes a sonic hedgehog (SHH) signal. For example, purmorphamine (9-cyclohexyl-N- [4- (4-morpholinyl) phenyl] -2- (1-naphthalenyloxy) -9H-purin-6-amine) useful for inducing ventralization Should be used. The concentration of purmorphamine (the amount added to the medium) is not particularly limited as long as the purpose of inducing differentiation along the neural cell lineage is achieved, but for example, 1 ng / ml to 5 μg / ml, preferably 10 to 500 ng / ml, more preferably 50 to 400 ng / ml. The optimum concentration can be set through preliminary experiments. SAG (N-methyl-N ′-(3-pyridinylbenzyl) -N ′-(3-chlorobenzo [b] thiophene-2-carbonyl) -1,4-diaminocyclohexane) is used as a hedgehog signal agonist. You can also The concentration (addition amount to the medium) when SAG is used is not particularly limited as long as the purpose of inducing differentiation along the neural cell lineage is achieved, but for example, 10 nM to 100 μM, preferably 100 nM to 10 μM, more preferably Is between 100 nM and 2 μM. The optimum concentration can be set through preliminary experiments.

  It is not essential that the concentration of each component (TGF-β family inhibitor, GSK3β inhibitor, FGF8 and hedgehog signal agonist) is constant throughout the entire culture period, and specific components (two or more components may be used) ) Or the concentration of all components may change during the culture. For example, the addition of FGF8 and hedgehog signal agonist is started on the second to sixth days of step (2). According to the said conditions, the rapid irritation | stimulation to a cell can be relieved. Preferably, the addition of FGF8 and hedgehog signal agonist is started on the third to fifth days of step (2).

  In order to promote differentiation induction along the neural cell lineage, a medium supplemented with leukemia inhibitory factor (LIF) is preferably used. As long as the purpose can be achieved, LIFs derived from various mammals can be used. However, it is preferable to match the origin (animal species) with the pluripotent stem cells to be used. Therefore, human LIF is preferably employed when human pluripotent stem cells are used. The concentration of LIF is not particularly limited, but is, for example, 0.25 ng / ml to 1 μg / ml, preferably 1 ng / ml to 50 ng / ml, more preferably 5 ng / ml to 20 ng / ml. The optimum concentration can be set through preliminary experiments.

  In order to promote differentiation induction along the neural cell lineage, a medium supplemented with bFGF (basic fibroblast growth factor) is preferably used. bFGF is also called FGF2. As long as the object of the present invention can be achieved, bFGF derived from various mammals can be used. However, it is preferable to match the origin (animal species) with the pluripotent stem cells to be used. Therefore, human bFGF is preferably employed when human pluripotent stem cells are used. Human FGF2 means that the human has the amino acid sequence of FGF2 that is naturally expressed in vivo. As a representative amino acid sequence of human FGF2, NP_001997.5 (fibroblast growth factor 2 [Homo sapiens]) can be exemplified by the NCBI accession number. The concentration of bFGF is not particularly limited, but is, for example, 0.25 ng / ml to 1 μg / ml, preferably 1 ng / ml to 50 ng / ml, more preferably 3 ng / ml to 30 ng / ml. The optimum concentration can be set through preliminary experiments.

  In order to suppress cell death, preferably, a medium to which a ROCK inhibitor (Rho-associated coiled-coil forming kinase / Rho-binding kinase) (for example, Y-27632 or Fasudil (HA-1077)) is also used is used. The concentration in the case of using Y-27632 as a ROCK inhibitor is, for example, about 1 μM to about 50 μM. The optimum concentration can be set through preliminary experiments.

  ROCK inhibitors strongly inhibit cell death when cells are in a dispersed state. Therefore, instead of using a ROCK inhibitor over the entire culture period of step (2), when seeding cells (ie, at the start of culture) or when collecting and dispersing cells for subculture, for example. Alternatively, the cells may be treated with a medium containing a ROCK inhibitor.

  Preferably, ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), fetal bovine serum so as to be advantageous for inducing differentiation along the neural cell lineage , Medium supplemented with N2 supplement, B27 supplement, etc. are used. The N2 supplement can be obtained from Gibco (product name N2 supplement (x100)) or the like, and the B27 supplement can be obtained from Gibco (product name B27 supplement (x100)) or the like.

  Furthermore, you may add another component to a culture medium as needed. Examples of components that can be added include insulin, iron sources (such as transferrin), minerals (such as sodium selenate), sugars (such as glucose), organic acids (such as pyruvic acid, lactic acid, etc.), serum proteins (such as albumin) Etc.), amino acids (eg L-glutamine etc.), reducing agents (eg 2-mercaptoethanol etc.), vitamins (eg ascorbic acid, d-biotin etc.), antibiotics (eg streptomycin, penicillin, gentamicin etc.), buffering agents (For example, HEPES).

  In this step (2), suspension culture is performed to form neurospheres. For example, serum-free agglutination suspension culture method (SFEB method / SFEBq method. Watanabe et al., Nature Neuroscience 8, 288-296 (2005), WO 2005/123902) and neurosphere method (Reynolds BA and Weiss S., Science, USA, 1992 Mar 27; 255 (5052): 1707-10) can be employed.

In the present invention, step (2) is carried out under normal oxygen partial pressure. In cell culture, conditions under which the oxygen concentration is lowered (low oxygen partial pressure / low oxygen concentration) may be used in consideration of the environment in the living body, and “normal oxygen partial pressure” in the present invention is like this. Contrast with special conditions. That is, “under normal oxygen partial pressure” is a condition in which the oxygen concentration is not intentionally adjusted. Although it may vary depending on other conditions (humidity, coexisting CO ₂ concentration, etc.), the oxygen concentration in the case of “normal oxygen partial pressure” is typically about 18% to about 22%.

Other culture conditions (incubation temperature, C0 ₂ concentration, etc.) can be appropriately set. The culture temperature is, for example, about 30 to 40 ° C, preferably about 37 ° C. The CO ₂ concentration is for example about 1-10%, preferably about 5%.

  The period (culture period) of step (2) is, for example, 7 to 21 days, preferably 10 to 16 days. If the culture period is too short or too long, the differentiation efficiency may be reduced.

  The formed neurospheres may be collected to dissociate the cells, and then subjected to further suspension culture. That is, subculture may be performed. However, the number of subcultures should be small, and the number of subcultures is preferably 1 or 0 (that is, no subculture is performed). The small number of subcultures is advantageous for the preparation of dopamine neurons in a short period of time, and is also effective in avoiding unintended differentiation induction (for example, induction of differentiation into glial cells). it is conceivable that. On the other hand, since subculture is effective in improving cell purity, it can be said that the subculture is optimally performed once. When subculture is performed once, the subculture is preferably performed on the 6th to 10th days from the start of step (2). In addition, when recovering neurospheres during subculture, it is preferable to prevent contamination of cells adhered to the surface of the incubator. Such an operation can contribute to the improvement of the preparation efficiency and purity of dopamine neurons.

In step (ii) following step (i), the neurospheres prepared in step (i) are transferred to neurons (eg, dopamine neurons, serotonin neurons, cerebral cortex excitatory neurons, inhibitory neurons, etc.). Culture under differentiation-inducing conditions. That is, in the present invention, the neurosphere itself is subjected to culture without separating and collecting cells from the neurosphere. This is one of the greatest features of the present invention, and by this, damage to cells associated with the separation / recovery operation (such as enzymatic treatment or physical treatment) can be avoided. As a result, stable calcium dynamics can be observed in a short time. Moreover, it is possible to evaluate in a state closer to a physiological environment / condition, which is advantageous in reproducing a state in which a differentiated nerve cell moves in a living brain. Therefore, the calcium dynamics evaluation method of the present invention is useful for capturing changes in calcium dynamics during development. In addition, culture media and culture conditions suitable for inducing differentiation into specific nerve cells such as dopamine neurons, serotonin neurons, cerebral cortex excitatory neurons, inhibitory neurons, etc. are known, and past reports and books, etc. Can be referred to. For example, regarding the differentiation induction into dopamine neurons, the basic culture method and operation can be referred to, for example, a protocol provided by ThermoFisher (published on the web page of ThermoFisher). Specifically, for example, differentiation into dopamine neurons is induced by adhesion culture in a medium containing a γ-secretase inhibitor, neurotrophic factor, ascorbic acid, TGF-β3, and cAMP or cAMP analog. Preferably, N- [N- (3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester is used as the γ-secretase inhibitor, and brain-derived neurotrophic factor (BDNF) is used as the neurotrophic factor. ) And glial cell-derived neurotrophic factor (GDNF), and diptyryl cAMP as a cAMP analog. Incidentally, the culture temperature, C0 although culture conditions ₂ concentration and the like can be appropriately set within a range suitable for the induction of differentiation to the target nerve cells, the culture temperature is, for example, about 30 to 40 ° C., preferably about 37 ° C., CO ₂ The concentration is for example about 1-10%, preferably about 5%.

  In step (iii), a calcium indicator is loaded on the migrated cells, and then the signal intensity from the calcium indicator is measured to examine changes in intracellular calcium. It is useful for elucidating the pathology of psychiatric / neurological diseases, etc., for example, to identify changes in intracellular calcium, screening for new drugs targeting psychiatric / neurological disorders that cause abnormal calcium dynamics, and existing drugs Alternatively, it can be used / applied for evaluating toxicity or teratogenicity of new drugs. The loading of the calcium indicator to the migrated cells may be performed by a conventional method. Typically, the culture solution is replaced with a solution containing a calcium indicator and incubated for a predetermined time (for example, 30 minutes to 1 hour). Thereafter, the solution is replaced with a measurement (recording) solution (for example, a medium or Tyrode), and the signal intensity from the calcium reagent is measured.

  For example, a fluorescent calcium indicator may be used as the calcium indicator. Various fluorescent calcium indicators have been developed. Examples of fluorescent calcium indicators include Fura 2-AM, Fura 2, Fluo 3-AM, Fluo 3, Fluo 4-AM, Fluo 4, Indo 1-AM, Indo 1, Rhod 2-AM, Rhod 2, Quin 2 (for example, Dojindo Laboratories, Inc., Thermo Fisher Scientific). Preferably, an acetoxymethyl (AM) ester fluorescent calcium indicator with high cell permeability is used. It should be noted that either one wavelength type fluorescent calcium reagent or two wavelength type fluorescent calcium reagent may be used.

  Kits for measuring intracellular calcium are also commercially available (for example, Calcium Kit-Fluo 4, Calcium Kit-Fra 2, Calcium Kit II-Fluo 4, Calcium Kit II-Fra 2, and Calcium Kit II from Dojindo Laboratories, Inc.). -Icelus, Thermo Fisher Scientific's Fluo-4 Imaging Kit, Rhod-3 Imaging Kit, DiscoverX Calcium No Wash Assay Kit), and such kits allow simple measurement of signal intensity. When a non-wash type kit is used, the signal intensity can be measured as it is (ie, without changing the solution / medium or washing operation) after loading the fluorescent calcium indicator.

  Measurement of the signal intensity from the calcium indicator may be performed using a device corresponding to the calcium indicator to be used. For example, when a fluorescent calcium indicator is used, a fluorescence measuring device (fluorescent plate reader, epifluorescence microscope, confocal microscope, etc.) may be used. For photographing / recording and analyzing the signal intensity, a photographing device (CCD camera, photodetector (photomultiplier tube, etc.)) or the like is used. Note that a dedicated system (for example, Infinite 200 series provided by Wako) has been developed for the measurement and analysis of intracellular calcium, and these systems may be used.

  The fluctuation of intracellular calcium is examined from the measurement result of the signal intensity. It is possible to examine intracellular calcium fluctuations (temporal changes) by performing at least two different measurements at the time of measurement. ) Perform measurements and capture the dynamics of intracellular calcium. According to the measurement over time, the number of occurrences and frequency of intracellular calcium concentration events, maximum rate of change, duration, etc., time constants and half-lives in the increase / decrease of intracellular calcium concentration, etc. Detailed information on intracellular calcium dynamics, such as the integrated value of the signal change rate, can be obtained. The “calcium event” refers to an event in which the signal intensity rises above a set threshold value.

  When performing measurement over time, the measurement time interval is set within a range of 0.1 seconds to 1 second, for example, and the measurement is continued for 5 minutes to 48 hours, for example.

  In the present invention, intracellular calcium is measured for at least one cell migrated from a neurosphere. Preferably, a plurality of cells (for example, 2 to 100 cells, preferably 5 to 50 cells) are measured. Increase the value, reliability, etc. of the evaluation by making it a measurement target and obtaining more data of the measurement results. As supported by the examples described later, cells that have migrated from neurospheres increase their differentiation and maturity with migration (migration). That is, it can be said that the migration distance reflects the differentiation / maturity of the cells, and the cells with the same migration distance have the same differentiation / maturity. Paying attention to this point, preferably, the intracellular calcium is measured for a plurality of cells having the same migration distance, and the measurement results are aggregated to examine the fluctuation of the intracellular calcium. In this way, not only can the reliability and objectivity of the evaluation results be improved, but also stable calcium dynamics in cells of a specific differentiation / maturity level can be evaluated. That is, this aspect is significant in that it enables an evaluation that cannot be realized by the conventional technique, that is, an evaluation of intracellular calcium dynamics in cells of a specific differentiation / maturity level.

A plurality of cells having the same migration distance can be specified (identified) based on the position of the cells, for example. Specifically, for example, it can be specified by the following method. First, the reference cell location radius 3 00μm (preferably a radius 200 [mu] m, more preferably a radius 100 [mu] m) with a focus of the cells present in the circle considered "migration distance equivalent cells". The “reference cell” can be arbitrarily specified. For example, a cell present at a distance of 500 μm to 1 cm from the outer edge of the neurosphere is defined as the “reference cell”.

In step (iii), if a signal intensity from a calcium indicator is measured after adding a substance capable of affecting intracellular calcium dynamics, the calcium dynamics in the presence of the substance can be evaluated. Such an evaluation system is useful as a means of examining mechanisms such as calcium influx and mobilization. The addition of “substances capable of affecting intracellular calcium kinetics” is usually performed after loading with a calcium indicator. “Substances that can affect intracellular calcium dynamics” include pathways involved in intracellular calcium dynamics (IP3 receptor-mediated pathway, ryanodine receptor-mediated pathway, VDCC (L-type, P-type, Q-type, N-type, R-type, or T-type))) (directly or indirectly) (referred to as “calcium pathway agonist” for convenience of explanation). Calcium pathway agonists such as promoters of IP3-dependent calcium mobilization (eg, ligands for IP3 receptors, IP3 receptor agonists) or inhibitors (eg, IP3 receptor antagonists), promoters of ryanodine-dependent calcium mobilization (eg, , Ligands of ryanodine receptors, ryanodine receptor agonists) or inhibitors (eg ryanodine receptor antagonists), promoters of VDCC-dependent calcium influx (eg L-type VDCC activators) or inhibitors (eg L-type VDCC inhibition) Agents), ligands for neurotransmitter receptors (dopamine receptors, GABA receptors, serotonin receptors, acetylcholine receptors, histamine receptors, etc.), activators (eg receptor agonists), inhibitors (eg receptors) Body antagonist), sodium channel inhibitor, potassium channel inhibitor, high concentration potassium It can be. Specific examples of calcium pathway agonists include TTX (sodium channel inhibitor), 2-APB (cell permeable allosteric inhibitor for IP3-induced Ca ²⁺ release), Nimodipine (L-type VDCC inhibitor), FPL 64176 ( Putative L-type VDCC activator), ATP, Suramin (non-selective P2Y receptor antagonist), Dantrolen (ryanodine receptor antagonist), histamine, serotonin, Dihydroxyphenylglycine (DHPG), GABA, glycine, dopamine, quinpirole (Quinpirole) D2 / D3 receptor agonist).

2. Drug Evaluation Method The second aspect of the present invention corresponds to the use or application of the calcium kinetic evaluation method of the present invention (first aspect), and is a drug evaluation method based on intracellular calcium kinetics of nerve cells (hereinafter, “ Referred to as “drug evaluation method of the present invention”). In the present specification, “drug evaluation” is used as a collective term for evaluation of drug efficacy and evaluation of toxicity. Therefore, in the present invention, the efficacy or toxicity of the test substance is evaluated. The term “toxicity” should be interpreted broadly, and in addition to general toxicity (acute toxicity, subacute toxicity, chronic toxicity), side effects, carcinogenicity, mutagenicity, teratogenicity, etc. are also toxicities. . Hereinafter, although the detail of the chemical | medical agent evaluation method of this invention is demonstrated, since it is the same as that of the said 1st aspect (calcium kinetics evaluation method of this invention) about the matter which is not mentioned especially, the description is abbreviate | omitted.

In the drug evaluation method of the present invention, the following steps (I) to (IV) are performed.
(I) a step of preparing a neurosphere in a culture vessel;
(II) culturing under conditions for inducing differentiation into nerve cells,
(III) loading the migrated cells with a calcium indicator;
(IV) adding a test substance;
(V) A step of measuring the signal intensity from the calcium indicator, examining changes in intracellular calcium, and evaluating the efficacy or toxicity of the test substance.

  Steps (I) and (II) are the same as steps (i) and (ii) of the calcium kinetic evaluation method of the present invention, respectively. The nerve cell in step (II) is a normal cell or a non-normal cell, and the term “normal cell” as used herein refers to a medicinal effect (improvement of a disease causing abnormal calcium dynamics, based on that) evaluated by the method of the present invention. It is a cell in which no abnormality is observed in relation to the therapeutic effect) or toxicity. A typical example of a normal cell is a cell derived from a healthy person, but, for example, suffers from a disease in which the medicinal effect of the evaluation target can exert a therapeutic effect, in other words, a disease that causes abnormal calcium dynamics (target disease). Cells from those who are not can also be used as normal cells. In the drug evaluation system of the present invention, when a patient-derived cell is used as a non-normal cell, in addition to the evaluation of drug efficacy for the purpose of screening (searching) for a therapeutic drug candidate, the toxicity of the therapeutic drug or therapeutic drug candidate Can also be evaluated (details will be described later). As described above, the method of the present invention has a unique feature that it can be used for both a drug efficacy evaluation system and a toxicity evaluation system by selecting cells to be used. Details of each evaluation system will be described later.

  The load of the calcium indicator in step (III) is in accordance with the description of step (iii) of the first aspect. After loading the calcium indicator, the test substance is added to form a state where the migrated cells and the test substance come into contact (step (IV)). The test substance is not particularly limited. Various substances that require evaluation of their efficacy or toxicity can be test substances. As the test substance, organic compounds or inorganic compounds having various molecular sizes can be used. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.) Existing or candidate ingredients such as pharmaceuticals, nutritional foods, food additives, agricultural chemicals, cosmetics (cosmetics) One of the preferable test substances is a plant extract, cell extract, culture supernatant, etc. The test substance may be used as a test substance by adding two or more kinds of test substances at the same time. It may be decided to examine the action, synergism, etc. The test substance may be derived from a natural product or synthesized, for example, in the latter case It is possible to build an efficient assay systems using techniques of combinatorial synthesis.

  In the next step (V), the signal intensity from the calcium indicator is measured, the fluctuation of intracellular calcium is examined, and the efficacy or toxicity of the test substance is evaluated. The signal intensity may be measured in the same manner as in the first aspect (calcium kinetic evaluation method). In addition, the analysis / analysis method and conditions of intracellular calcium fluctuations using the signal intensity measurement results also conform to the first aspect.

  As described above, the drug evaluation method of the present invention can be used as a drug efficacy evaluation system or a toxicity evaluation system. In the former case, the efficacy of the test substance is determined in this step, and in the latter case, this step. The toxicity of the test substance is determined. In the present invention, “change in calcium dynamics” is used as an index of drug efficacy or toxicity. When configured as a medicinal effect evaluation system, typically, the nerve cell in step (II) is a non-normal cell, and normalization of calcium kinetics (ie, approaching calcium kinetics of normal cells) by addition of a test substance is performed. When it is confirmed, the test substance is determined to be effective (medicinal). Further, since the degree of normalization of calcium dynamics reflects the degree of effectiveness (drug efficacy), the degree or strength of drug efficacy can also be determined based on the degree of normalization. Test substances that have been validated can be candidates for drugs that target calcium dynamics. According to the medicinal effect evaluation system, it is possible to screen for a drug component effective for various diseases (target diseases) that cause abnormal intracellular calcium dynamics or a candidate thereof.

  When configured as a toxicity evaluation system, the nerve cells in step (II) are normal cells or non-normal cells. In the case of normal cells, the test substance is determined to be toxic when a change in calcium dynamics is observed by the addition of the test substance. In addition, since the degree of change in calcium dynamics reflects the degree of toxicity, the degree or intensity of toxicity can be determined based on the degree of change. On the other hand, in the case of non-normal cells, it is determined that the test substance is toxic when a further abnormality in calcium dynamics is observed by the addition of the test substance. Further abnormalization is that the difference from normal calcium kinetics (ie, normal cell calcium kinetics) becomes more prominent. According to the toxicity evaluation system, whether or not a test substance such as a pharmaceutical ingredient or candidate, a dietary supplement (supplement) or candidate, a food additive or candidate thereof shows toxicity by affecting intracellular calcium dynamics No or the degree of toxicity can be assessed.

  In one aspect (first aspect) of the present invention, the signal intensity is measured before and after the addition of the test substance (step (IV)), and the measurement result before the addition of the test substance is compared with the measurement result after the addition. Is done. Based on the comparison result, the efficacy or toxicity of the test substance is evaluated. According to this aspect, since the calcium dynamics in the absence and presence of the test substance can be evaluated using the same cell, there is an advantage that the heterogeneity between the cells of the calcium indicator does not affect the interpretation. is there.

  In another embodiment (second embodiment), the signal intensity is measured after the addition of the test substance (step (IV)), and the change in intracellular calcium after the addition of the test substance is examined. In this embodiment, in principle, a cell ("control") treated under the same conditions except that the test substance is not added is prepared, and based on a comparison with the measurement result (variation of intracellular calcium) for the cell. The efficacy or toxicity of the test substance is evaluated.

  Before adding the test substance, pathways involved in intracellular calcium dynamics (IP3 receptor-mediated pathway, ryanodine receptor-mediated pathway, VDCC (L-type, P-type, Q-type, N-type, R-type or Measurement of signal intensity before and after addition of test substance (ie, measurement similar to the first aspect) by adding a substance (calcium pathway agonist) that directly or indirectly acts on the pathway via T type) Alternatively, by measuring the signal intensity after the addition of the test substance (that is, the same measurement as in the second embodiment), whether or not the test substance affects the action of the calcium pathway agonist and / or the degree thereof is examined ( Third aspect). The “calcium pathway acting drug” here is as described in the first aspect (calcium kinetic evaluation method). This aspect is particularly characterized in that useful information regarding the mechanism of action of the test substance (ie which route the test substance targets) is obtained. In this embodiment, in particular, a substance that has been found to affect calcium kinetics (for example, a substance that has been confirmed to be effective or toxic by the evaluation method of the first aspect or the second aspect) is adopted as a test substance. Good.

  Hereinafter, substances targeting the IP3 receptor pathway (ie, promoters or inhibitors of IP3-dependent calcium mobilization) or substances targeting the L-type VDCC pathway (ie, promoters of L-type VDCC-dependent calcium influx) (Or an inhibitor) as a “calcium pathway agonist”, useful information provided when the third aspect is a medicinal effect evaluation system will be described.

(1) When the test substance attenuates or abolishes the action of the promoter of IP3-dependent calcium mobilization The test substance can be expected to have a medicinal effect by inhibiting or suppressing calcium mobilization via the IP3 receptor, and accepting IP3 The test substance is promising as a drug or a seed compound for diseases caused by excessive mobilization of calcium through the body, for example, Huntington's disease, spinocerebellar degeneration 2 and 3, Alzheimer's disease.

(2) When the test substance attenuates or abolishes the action of an inhibitor of IP3-dependent calcium mobilization The test substance can be expected to have a medicinal effect by promoting calcium mobilization via the IP3 receptor. The test substance is promising as a drug or a seed compound for diseases caused by insufficient mobilization of calcium, such as spinocerebellar degeneration types 15 and 16.

(3) When the test substance attenuates or invalidates the action of the promoter of L-type VDCC-dependent calcium influx The test substance can be expected to have a medicinal effect by inhibiting or suppressing calcium mobilization via L-type VDCC, The test substance is promising as a drug or seed compound for diseases caused by excessive mobilization of calcium via L-type VDCC, such as Timothy syndrome, Parkinson's disease.

(4) When the test substance attenuates or nullifies the action of an inhibitor of L-type VDCC-dependent calcium influx The test substance can be expected to have a medicinal effect by promoting calcium mobilization via L-type VDCC, and L-type The test substance is promising as a drug or a seed compound for diseases caused by lack of calcium mobilization via VDCC, for example, Brugada syndrome.

  Even when the third aspect is a toxicity evaluation system, as in the case described above, it becomes clear which of the plurality of pathways involved in intracellular calcium dynamics is targeted by the test substance. It is possible to evaluate toxicity including the introduction.

  In any of the first aspect, the second aspect, and the third aspect, preferably, the signal intensity is measured over time, and the measurement result (change in intracellular calcium over time) is used for evaluation of drug efficacy or toxicity. . According to the measurement over time, the number of occurrences and frequency of intracellular calcium concentration events, maximum rate of change, duration, etc., time constants and half-lives in the increase / decrease of intracellular calcium concentration, etc. Detailed information on intracellular calcium dynamics, such as the integrated value of the signal change rate, can be obtained. When performing such measurement over time, the measurement time interval is set within a range of 0.1 to 1 second, for example, and the measurement is continued for 5 minutes to 48 hours, for example. Although measurement over time is preferable in the present invention, in the second and third aspects, at a specific time after the addition of the test substance (for example, the measurement time is set within a range of 5 seconds to 24 hours after the drug is added). It is also possible to measure the signal intensity and evaluate the efficacy or toxicity of the test substance based on comparison with a reference signal intensity (typically, the signal intensity of the control).

  By the way, as shown in Examples described later, in cells that migrated when neurospheres were cultured under conditions for inducing differentiation into dopamine neurons, the main calcium source was changed from IP3 receptor-dependent calcium mobilization to L-type VDCC-dependent calcium. Changed to inflow. Based on this finding, preferably, the induction of differentiation into dopamine neurons in step (II) allows migration of dopamine neurons from neurospheres, and the promotion of IP3-dependent calcium mobilization as described above. An agent or an inhibitor is added, and the signal intensity of step (V) is measured between the start of culture under differentiation-inducing conditions and the seventh day (Aspect (A)), or under differentiation-inducing conditions. The signal intensity of step (V) is measured during the period from the 14th day to the 30th day after the start of the culture (Aspect (B)). According to the aspect (A), when the main calcium source in dopamine neurons is IP3-dependent calcium mobilization, the action / effect of the test substance on the mobilization (in other words, the IP3 receptor pathway) can be evaluated. On the other hand, according to the embodiment of (B), when the main calcium source in dopamine neurons is L-type VDCC-dependent calcium influx, the action / effect of the test substance on the mobilization (in other words, L-type VDCC pathway) The effect can be evaluated, and in any case, more useful information can be obtained.

  The following experiments were conducted with the aim of constructing a new assay system that enables observation and evaluation of stable calcium dynamics in nerve cells.

1. Method (1) Culture of neurospheres iPS cells derived from healthy individuals were cultured in iPS cell medium supplemented with SB431542 (3 μM), CHIR99021 (3 μM), and dorsomorphin (3 μM) for 7 days (Day 0 to Day 7). . After that, disperse by TrypLE ^™ select (Thermo Fisher Scientific Inc.) and pass through cell strainer. Neurosphere medium (DMEM / F12 supplemented with 1 × N2 supplement, 0.6% glucose, penicillin / streptomycin, 5 mM HEPES) (MHM medium) supplemented with 1 × B27 supplement, 20 ng / ml bFGF, 10 ng / ml human LIF, 10 μM Y27632, 3 μM CHIR99021, 2 μM SB431542, 100 ng / ml FGF8 and 1 μM purmorphamine Neurospheres were formed by suspension culture for 2 weeks (7th to 21st days). FGF8 and purmorphamine were added from the 10th day. On the 14th day, the neurospheres were collected and dispersed to form single cells, and then cultured again in suspension to re-form neurospheres (secondary neurospheres).

(2) Differentiation into dopaminergic neurons Next, the above neurospheres were differentiated into dopaminergic cells by the following method. Dopaminergic neuron differentiation differentiation medium (B27 supplement to MHM medium, 10 μM DAPT, 20 ng / ml BDNF, 20 ng /) on 35 mm diameter glass bottom dish (glass surface diameter 1 mm) coated with matrigel (1 to 3.3%) 1.6 ml of GDNF, 0.2 mM ascorbic acid, 1 ng / ml TGF-β3 and 0.5 mM dbcAMP) was added, and neurospheres were added to the medium and cultured in a CO ₂ incubator. During the culture, the medium for inducing differentiation of dopaminergic neurons was exchanged 0.8 ml twice a week.

(3) Calcium imaging Calcium imaging was performed using juvenile neurons 7 to 17 days after initiation of differentiation induction into dopaminergic neurons. The entire amount of dopaminergic neuron differentiation induction medium was recovered from the culture dish during culture and stored as a conditioned medium. A dopaminergic neuron differentiation differentiation medium supplemented with 5 μM Fluo4-AM (Thermo) was added to the culture dish and allowed to stand at 37 ° C. for 30 minutes to incorporate Fluo4-AM into young neurons. After uptake of Fluo4-AM, the medium was removed, the stored conditioned medium was added, and the juvenile nerve cells were allowed to stand at 37 ° C. for 30 minutes. After that, immature neurons were placed in a small CO ₂ incubator on the inverted confocal microscope LSM710 (Zeiss) stage, and calcium imaging was performed on cells that were more than 100 μm away from the outer periphery of the cell aggregate that was a neurosphere. It was. Imaging was performed using a 20x / NA-0.8 objective lens for 20 to 60 minutes at a frequency of 0.5 to 1.0 Hz (acquired image was 512 × 512 pixels). Fluo-4 was excited with an Argon laser (488 nm), and fluorescence was detected with a QUASAR detector. 50 μM 2-APB (FIGS. 1 and 2), 1 μM or 3 μM TTX (FIG. 3), 10 μM Nimodipine (FIG. 4), and 5 μM FPL 641765 (FIG. 4) were added by a self-made drug adder during imaging.

(4) Quantitative analysis of calcium imaging Image analysis was performed by ImageJ (NIH). Create a superimposed image in the time direction from a series of Fluo-4 images captured over time, and after binarizing this image, split the image with Watershed and extract ROIs (Region of interests) (See FIG. 6). ROIs were applied to a series of time-lapse images, and changes in Fluo-4 luminance values over time were measured for each cell. The Fluo-4 brightness value for each frame is calculated by moving the median value (61 frame width) for the lower 25% after moving average processing (5 frame width) after subtracting the background value calculated from the cell-free area. The Fluo-4 luminance value after moving average processing was standardized by the obtained trace to obtain an F value. Further, the lower 25% value was extracted from the series of F values, the average value was defined as F0, and ΔF / F0 = (F−F0) / F0 was calculated for each frame. In addition, the standard deviation (SD) of the F value lower 25% was calculated, and the ΔF / F0 apex of F0 + 7SD or more was defined as a calcium event (see FIG. 7, the peak is indicated by a circle). Furthermore, the detected ΔF / F0 vertex and the ΔF / F0 period of F0 + 2SD or more were defined as the calcium event occurrence period (filled in FIG. 7). Details of the waveform analysis are shown in FIG.

2. Results The results of calcium imaging are shown in FIGS. By adding 2-APB, an inhibitor of IP3-dependent calcium mobilization, to the young neurons on the 8th day after the initiation of differentiation induction, fluctuations in the amount of intracellular calcium were suppressed (FIG. 1 right). On the other hand, when 2-APB was added on the 14th day after the initiation of differentiation induction, the change in the amount of intracellular calcium was only slightly affected (FIG. 2).

  In addition, as a result of examining the response to TTX, which is a sodium channel inhibitor, intracellular calcium dynamics were not affected even when added on day 7 after the initiation of differentiation induction (results not shown), whereas on day 14 When added to the cell, the fluctuation of the amount of intracellular calcium was suppressed (inflow of calcium into the cell was strongly inhibited) (FIG. 3).

  When Nimodipine, an inhibitor of L-type VDCC-dependent calcium influx, was added 15 days after the start of differentiation induction, fluctuations in intracellular calcium were suppressed (Fig. 4, left), and a promoter of L-type VDCC-dependent calcium influx. When a certain FPL64176 was added on the 17th day after initiation of differentiation induction, the fluctuation of intracellular calcium amount increased (FIG. 4 right).

  From this, it can be seen that the calcium fluctuation at this time is due to L-type VDCC-dependent calcium constituted by, for example, CACNA1C or CACNA1D in the brain.

  As shown by the above results, the intracellular calcium dynamics could be observed with high stability and accuracy despite the short-time measurement. In addition, changes in molecular pathways contributing to calcium dynamics (ie, transition from IP3 receptor pathway to L-type VDCC pathway) could be captured. The experimental results are summarized in FIG.

  The calcium kinetics evaluation method of the present invention is useful for evaluating intracellular calcium kinetics of various nerve cells. According to the present invention, it is possible to stably perform force lucium imaging reflecting the function of nerve cells in a short period of time. In addition, it is possible to evaluate the drug efficacy or toxicity focusing on the influence on the intracellular calcium kinetics (change in calcium kinetics) important in the survival, proliferation, maturation, etc. of nerve cells.

  The culture system used for the present invention (culturing neurospheres under conditions for inducing differentiation into neurons) mimics the developmental process. Because of this feature, according to the present invention, it is possible to evaluate calcium dynamics in cells undergoing differentiation and maturation, evaluation of calcium dynamics based on the molecular basis peculiar to developmental stage and cell type, teratogenicity evaluation, differentiation process of newborn neurons It is possible to evaluate the impact on

  Targeting psychiatric / neurological disorders that lead to abnormalities in calcium dynamics (eg Parkinson's disease, anxiety disorders, drug dependence) The application or application of the present invention to the screening of new drugs (evaluation of drug efficacy), the evaluation of toxicity or teratogenicity of existing drugs or new drugs, etc. is envisaged.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (20)

  A method for evaluating calcium dynamics in nerve cells, characterized by measuring intracellular calcium in neurons migrating from neurospheres. The method for evaluating calcium kinetics according to claim 1, comprising the following steps (i) to (iii):
(I) preparing a neurosphere in a culture vessel;
(Ii) culturing under conditions for inducing differentiation into nerve cells,
(Iii) A step of measuring the intracellular calcium fluctuation by measuring the signal intensity from the calcium indicator after loading the migrated cells with the calcium indicator.   The method for evaluating calcium kinetics according to claim 1 or 2, wherein the neurosphere is derived from a pluripotent stem cell.   The method for evaluating calcium kinetics according to claim 3, wherein the pluripotent stem cell is an induced pluripotent stem cell.   The calcium dynamics evaluation method according to any one of claims 1 to 4, wherein the cells constituting the neurosphere are non-normal cells.   The method for evaluating calcium dynamics according to claim 5, wherein the non-normal cell is a disease cell having a genetic characteristic of a target disease.   The method for evaluating calcium kinetics according to claim 6, wherein the diseased cell is a patient-derived cell or a cell created by genetic manipulation.   The calcium dynamics evaluation method according to any one of claims 1 to 7, wherein the nerve cell is a dopamine nerve cell.   The calcium dynamics evaluation method according to any one of claims 1 to 8, wherein intracellular calcium is measured over time. The calcium kinetics evaluation method according to any one of claims 1 to 9, wherein the neurosphere is prepared by a method comprising the following steps (1) and (2):
(1) culturing pluripotent stem cells in the presence of a TGF-β family inhibitor, a GSK3β inhibitor and a BMP inhibitor;
(2) A step of subjecting the cells obtained in step (1) to suspension culture in the presence of a TGF-β family inhibitor, GSK3β inhibitor, FGF8 and hedgehog signal agonist and normal oxygen partial pressure to form neurospheres .   In step (iii), intracellular calcium is measured for a plurality of cells having the same migration distance, and the measurement results are aggregated to evaluate fluctuations in intracellular calcium. Calcium dynamics evaluation method.   In step (iii), the number of occurrences or frequency of the intracellular calcium concentration increase event, the maximum rate of change, or the duration, the time constant or half-life in the increase or decrease of the intracellular calcium concentration, and the signal in the intracellular calcium concentration increase event The method for evaluating calcium kinetics according to any one of claims 2 to 10, wherein a change in intracellular calcium is examined based on one or more items selected from the group consisting of an integral value of a change rate.   The signal intensity from a calcium indicator is measured after adding a substance that acts directly or indirectly on a pathway involved in intracellular calcium dynamics in step (iii). Calcium dynamics evaluation method. Drug evaluation method comprising the following steps (I) to (III):
(I) a step of preparing a neurosphere in a culture vessel;
(II) culturing under conditions for inducing differentiation into nerve cells,
(III) loading the migrated cells with a calcium indicator;
(IV) adding a test substance;
(V) A step of measuring the signal intensity from the calcium indicator, examining changes in intracellular calcium, and evaluating the efficacy or toxicity of the test substance.   In step (V), the signal intensity is measured before and after addition of the test substance, and the measurement result before addition of the test substance and the measurement result after addition of the test substance are compared, and the efficacy or toxicity of the test substance is evaluated. The drug evaluation method according to claim 14.   In step (V), the measurement result of signal intensity is compared with the measurement result of signal intensity of cells treated under the same conditions except that the test substance is not added, and the efficacy or toxicity of the test substance is evaluated. Item 15. The drug evaluation method according to Item 14.   The method for evaluating a drug according to any one of claims 14 to 16, wherein a substance that directly or indirectly acts on a pathway involved in intracellular calcium dynamics is added before the test substance is added.   18. The agent of claim 17, wherein the substance is a promoter or inhibitor of inositol triphosphate (IP3) -dependent calcium mobilization, or a promoter or inhibitor of L-type voltage-dependent calcium channel (VDCC) -dependent calcium influx. Drug evaluation method.   The drug evaluation method according to claim 18, wherein the nerve cell is a dopamine neuron, and the signal intensity of step (V) is measured between the start of culture under differentiation-inducing conditions and the seventh day.   19. The method for evaluating a drug according to claim 18, wherein the nerve cell is a dopamine neuron, and the signal intensity of step (V) is measured from the 14th day to the 30th day after the start of culture under differentiation-inducing conditions. .

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