WO2019107481A1 - Method for evaluating cell hyperstimulation toxicity - Google Patents

Abstract

This method for evaluating hyperstimulation toxicity to a human neurocyte of a test substance comprises: (1) a step for bringing a test substance into contact with a human neurocyte prepared by inducing differentiation from pluripotent stem cells; (2) a step for imparting a stimulation to the human neurocyte; (3) a step for measuring the degree of hyperstimulation of the human neurocyte; and (4) a step for evaluating that the test substance has hyperstimulation toxicity to a human neurocyte when the hyperstimulation of the human neurocyte is enhanced in the presence of the test substance.

Description

Cell excitotoxicity evaluation method

The present invention relates to a method of evaluating excitotoxicity to human neurons.
Priority is claimed on Japanese Patent Application No. 2017-230610, filed Nov. 30, 2017, the content of which is incorporated herein by reference.

By the time of clinical application of therapeutic drug candidate substances, many of them are unplanned side effects due to unexpected side effects. In particular, unlike in humans, side effects are often overlooked in preclinical studies using model animals whose neurological symptoms are difficult to assess. Therefore, there is a need to predict nervous system side effects that are often exposed for the first time in human phase I or phase II clinical trials and incorporate them into a therapeutic drug development scheme.

However, in vitro side effect prediction of nervous system side effects has hardly been performed due to instability of the cell source used and lack of an appropriate evaluation method.

As a method of predicting side effects of nervous system side effects, it is known that nerve cell excitability is evaluated by measuring an increase in intracellular calcium concentration using nerve cells prepared from rat or guinea pig (non-patented) Document 1, non-patent document 2). In addition, Patent Document 1 discloses a method of rapidly and synchronously differentiating pluripotent stem cells into neurons.

International Publication No. 2014/148646

However, in the evaluation methods of Non-Patent Documents 1 and 2, neural cells are prepared from animals, and therefore, evaluation methods using human nerve cells are not used. Moreover, the said evaluation method has a problem also in the stable supply of a cell, and is not suitable for high-throughput evaluation. In addition, it has not been examined whether or not the nerve cells induced to differentiate by the method described in Patent Document 1 can be applied to the evaluation of nerve cell excitotoxicity. Under such circumstances, there is a need for a method for evaluating the excitotoxicity of a test substance to nerve cells that can predict in vitro nervous system side effects, which can respond to stable and high-throughput evaluation. Therefore, an object of the present invention is to provide a method for evaluating excitotoxicity of a test substance to human neurons, which can respond to stable and high-throughput evaluation.

The present invention includes the following aspects.
[1] A method for evaluating the excitotoxicity of a test substance to human neurons,
(1) bringing a test substance into contact with human neurons produced by inducing differentiation of pluripotent stem cells;
(2) a step of stimulating the human neural cell,
(3) measuring the degree of excitation of the human nerve cell, and (4) if the excitation of the human nerve cell is enhanced in the presence of the test substance, the test substance is excitotoxic to the human nerve cell A method for evaluating excitotoxicity of a test substance on human neurons, which comprises the step of evaluating the presence of the compound.
[2] The method according to [1], wherein the human neural cell is a cell that has been passaged once after differentiating pluripotent stem cells into neural cells.
[3] The method according to [1] or [2], wherein the stimulation to human neurons is an electrical stimulation.
[4] The method according to [3], wherein the electrical stimulation is performed at a voltage of 10 to 15 V and a stimulation frequency of 8 to 30 Hz.
[5] The method according to any one of [1] to [4], wherein the step of measuring the degree of excitation of the human nerve cell is the measurement of calcium dynamics in the human nerve cell.
[6] The method according to any one of [1] to [5], wherein the human neuronal cell is a cerebral cortical neuronal cell.

According to the present invention, it is possible to provide a method for evaluating the excitotoxicity of a test substance on human neural cells, which can predict in vitro nervous system side effects that can respond to stable and high-throughput evaluation.

In Example 1, it is the figure which showed the change of the intracellular calcium at the time of Bepridil addition. In Example 2, it is the figure which showed the change of the intracellular calcium at the time of adding Amoxapin, Chlorpromazine, and Linopirdin, respectively.

In one embodiment, the present invention is a method for evaluating excitotoxicity of a test substance on human neurons,
(1) bringing a test substance into contact with human neurons produced by inducing differentiation of pluripotent stem cells;
(2) a step of stimulating the human neural cell,
(3) measuring the degree of excitation of the human nerve cell, and (4) if the excitation of the human nerve cell is enhanced in the presence of the test substance, the test substance is excitotoxic to the human nerve cell The present invention provides a method for evaluating the excitotoxicity of a test substance to human neurons, which comprises the step of evaluating the presence.
The present embodiment will be described in detail below.

Human neural cells used in the method for evaluating excitotoxicity of the present embodiment can be produced from pluripotent stem cells by differentiation induction according to the method described in Patent Document 1. Specifically, human neural cells can be prepared from pluripotent stem cells as follows. In the following description, a neuron used in the method of evaluating excitotoxicity according to the present invention may be called iN in order to distinguish it from an original (that is, natural) neuron. In addition, the Ngn2 gene may be referred to as a neurogenic factor (or N-factor).

<Method of producing neural cells from pluripotent stem cells>
A nucleic acid encoding Ngn2 is introduced into pluripotent stem cells using a transposon, and the expression of the gene can be maintained for 3 days or more to allow rapid and synchronized differentiation into neural cells. The expression induction of the Ngn2 gene can be performed either in culture or in an animal.
Below, the technology which comprises these methods is explained in full detail.

<Pluripotent stem cells>
The pluripotent stem cells used in the method for evaluating excitotoxicity according to the present embodiment are stem cells having pluripotency capable of differentiating into all cells present in a living body and also having proliferation ability. . Examples include, but are not limited to, embryonic stem (ES) cells, cloned embryonic derived embryonic stem (ntES) cells obtained by nuclear transfer, spermatogonia stem cells (GS cells), embryonic germ cells (EG) Cells), induced pluripotent stem (iPS) cells, cultured fibroblasts and pluripotent cells derived from bone marrow stem cells (Muse cells). Among these, pluripotent stem cells preferable for use in the excitotoxicity evaluation method of the present embodiment are ES cells, nt ES cells, and iPS cells. Each stem cell is described below.

(A) Embryonic Stem Cell ES cell is a stem cell having pluripotency and proliferation ability by self-replication established from the inner cell mass of a mammalian early embryo (eg, blastocyst) such as human and mouse.
ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, which is the 8-cell stage of fertilized eggs and embryos after morula, and have the ability to differentiate into any cells that constitute an adult, so-called highly differentiated cells. It has the ability to grow by self-replication.

Human ES cell lines such as WA01 (H1) and WA09 (H9) are obtained from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from the Research Institute for Regenerative Medicine (Kyoto, Japan) It is possible.

(B) Sperm Stem Cells Sperm stem cells are testis-derived pluripotent stem cells, and are cells serving as a source for spermatogenesis. These cells, like ES cells, can be induced to differentiate into cells of various lineages, and for example, have the property of being able to produce chimeric mice when transplanted into mouse blastocysts. It is capable of self-replication in a culture solution containing glial cell line-derived neurotrophic factor (GDNF), and spermatozoa by repeating passaging under culture conditions similar to ES cells. Stem cells can be obtained.

(C) Embryonic Germ Cells Embryonic germ cells are cells with pluripotency similar to ES cells, established from embryonic primordial germ cells, such as LIF, bFGF, stem cell factor, etc. It can be established by culturing primordial germ cells in the presence of

(D) Artificial pluripotent stem cells Artificial pluripotent stem (iPS) cells can be prepared by introducing specific reprogramming factors in the form of DNA or protein into somatic cells, and are almost equivalent to ES cells Artificial stem cells derived from somatic cells, which have the following characteristics: pluripotency of differentiation and proliferation ability by self-replication. The reprogramming factor is a gene specifically expressed in ES cells, its gene product or non-cording RNA, or a gene that plays an important role in the maintenance of undifferentiated ES cells, its gene product or non-cording RNA, or You may be comprised by the low molecular weight compound. As genes included in the reprogramming factor, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 etc. are exemplified, and these reprogramming factors may be used alone or in combination.

The reprogramming factors include histone deacetylase (HDAC) inhibitors [eg, valproic acid (VPA), trichostatin A, sodium butyrate, small molecule inhibitors such as MC 1293, M344, siRNA for HDAC and shRNA (eg, Nucleic acid expression inhibitors such as HDAC1 siRNA Smartpool (trademark) (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.], MEK inhibitors (eg PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen Synthase kinase-3 inhibitor (eg, Bio and CHIR99021), DNA methyltransferase inhibitor For example, 5-azacytidine), histone methyltransferase inhibitors (for example, small molecule inhibitors such as BIX-01294, nucleic acid expression inhibitors such as Suv39hl, Suv39h2, SetDB1 and G9a, nucleic acid expression inhibitors such as shRNA, etc.), L-channel calcium agonists (eg, Bayk 8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (eg, LY364947, SB431542, 616453 and A-83-01), p53 inhibitors (eg, siRNA and shRNA against p53), ARID3A inhibitors (eg, ARID3A) , SiRNA for ARID 3 A and shRNA), miRNA such as miR-291-3p, miR-294, miR-295 and mir-302, Wn Increase the establishment efficiency of t Signaling (eg soluble Wnt3a), neuropeptide Y, prostaglandins (eg prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLIS1, PITX2, DMRTB1 etc The factors used for the purpose are also included, and in the present specification, the factors used for the purpose of improving the establishment efficiency are also not distinguished from the reprogramming factors.

The reprogramming factor may be introduced into somatic cells in the form of a protein, for example, by lipofection, fusion with a cell membrane permeable peptide (eg, TAT and polyarginine derived from HIV), microinjection and the like.

On the other hand, in the case of the form of DNA, it can be introduced into somatic cells by techniques such as viruses, plasmids, vectors such as artificial chromosomes, lipofection, liposomes, microinjection and the like.

In the case of the form of RNA, for example, it may be introduced into somatic cells by a technique such as lipofection, microinjection or the like, and RNA in which 5-methylcytidine and pseudouridine have been incorporated may be used to suppress degradation.

As a culture medium for iPS cell induction, for example, DMEM, DMEM / F12 or DME culture medium containing 10 to 15% FBS (LIF, penicillin / streptomycin, puromycin, L-glutamine are further added to these cultures. , Non-essential amino acids, β-mercaptoethanol, etc. can be suitably included) or commercially available culture fluid [eg, culture fluid for culturing mouse ES cell (TX-WES culture fluid, Thrombox X), primate ES cell Culture media (culture fluid for primate ES / iPS cells, Reprocel), serum-free medium (mTESR, Stemcell Technology), and the like are included.

As an example of the culture method, for example, a somatic cell is contacted with a reprogramming factor on DMEM or DMEM / F12 culture medium at 10% FBS in the presence of 5% CO ₂ at 37 ° C. and cultured for about 4 to 7 days The cells are then replated on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.), and after about 10 days after contact of somatic cells with the reprogramming factor, using a culture medium for culturing bFGF-containing primate ES cells An iPS-like colony can be generated after about 30 to about 45 days or more from the contacting and culturing.

Alternatively, DMEM culture solution containing 10% FBS on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.) at 37 ° C. in the presence of 5% CO ₂ (in addition, LIF, penicillin / streptomycin, Puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc. may optionally be included) and allowed to form ES-like colonies after about 25 to about 30 days or more . Desirably, a method using a somatic cell to be initialized itself or an extracellular matrix (for example, Laminin-5 and Matrigel (BD)) instead of feeder cells is exemplified.

Besides this, a method of culturing using a medium not containing serum is also exemplified. Furthermore, in order to increase the establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less).
During the above culture, culture medium exchange with fresh culture medium is performed once daily from the second day after the culture start. Also, the number of somatic cells used for nuclear reprogramming is not limited, but is in the range of about 5 × 10 ³ to about 5 × 10 ⁶ cells per 100 cm ^{2 of} culture dish.

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

As used herein, the term "somatic cell" refers to any animal cell except germline cells such as oocytes, oocytes and ES cells or totipotent cells (preferably mammalian cells including human) Say). Somatic cells include, but are not limited to, fetal (child) somatic cells, neonatal (child) somatic cells, and any mature healthy or diseased somatic cells, and also primary culture cells. Also included are passage cells and 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, dental pulp stem cells, (2) tissue precursor cells, (3) lymphocytes, epithelium Cells, endothelial cells, muscle cells, fibroblasts (skin cells etc.), hair cells, hepatocytes, gastric mucous cells, enterocytes, spleen cells, pancreatic cells (pancreatic exocrine cells etc.), brain cells, lung cells, kidney cells And differentiated cells such as adipocytes.

Moreover, from the viewpoint of producing a model cell of a pathological condition, a diseased somatic cell may be used. Here, the disease is exemplified by neurodegenerative diseases. In the case of producing a model cell of a pathological condition using the method for producing neural cells described above, iPS cells may be produced using somatic cells derived from patients with neurodegenerative diseases. Here, "neurodegenerative disease" is a disease caused by degeneration or loss of nerve cells, and exemplified by Alzheimer's disease, Parkinson's disease, Lewy body disease, Huntington's disease, spinocerebellar degeneration, etc. Be done. For example, somatic cells of patients with Alzheimer's disease are exemplified by somatic cells having mutations in the presenilin 1 gene or the preselinin 2 gene, and more than 30 mutations have been reported for presenilin 1 mutations so far, for example, It includes mutations in which D257 or D385 is substituted with alanine or glutamic acid, but is not particularly limited thereto.

(E) Cloned Embryo-Derived ES Cells Obtained by Nuclear Transfer nt ES cells are cloned embryo-derived ES cells prepared by nuclear transfer technology and have almost the same characteristics as fertilized egg-derived ES cells There is. That is, ES cells established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing unfertilized egg nuclei with somatic cell nuclei are nt ES (nuclear transfer ES) cells. For the generation of nt ES cells, a combination of nuclear transfer technology and ES cell generation technology (described above) is used. In nuclear transfer, the nucleus of a somatic cell can be injected into an enucleated unfertilized egg of a mammal, and reprogramming can be performed by culturing for several hours.

(F) Multilineage-differentiating Stress Enduring cells (Muse cells)
Muse cells are pluripotent cells obtained by subjecting fibroblasts or bone marrow stromal cells to trypsin treatment for a long time, preferably for 8 hours or 16 hours, and then suspension culture; And CD105 is positive.

In the excitotoxic evaluation method of the present embodiment, pluripotent stem cells into which a gene causing a disease is introduced can be used from the viewpoint of producing a model cell of a pathological condition. Examples of pluripotent stem cells having a mutated gene include iPS cells produced by isolating somatic cells of patients with Alzheimer's disease and the like.
In the pluripotent stem cells used in the method for evaluating excitotoxicity according to the present embodiment, the Alzheimer's disease model cells of dementia include nerve cells obtained from the pluripotent stem cells by the method described above. In pluripotent stem cells used in the excitotoxic evaluation method of the present embodiment, model cells of a preferable pathological condition are human cells.

<Neural cell>
In the excitotoxicity evaluation method of the present embodiment, a neuron is defined as a cell that expresses one or more marker genes of a neuron such as β-III tubulin, NCAM, and MAP2 and has a neurite. Thus, the criteria for the neural cells (ie, iN) produced by the method according to the invention also follow this. Furthermore, the neurons produced in the present invention are preferably glutamatergic. And, in the present invention, producing nerve cells means obtaining a cell population containing cells satisfying the above definition, preferably, 50%, 60%, 70%, 80% or 90% of the cells. It is to obtain a cell population containing the above.
Since Tuj1 is an anti-β-III tubulin, cells expressing the β-III tubulin may be referred to as Tuj1-positive cells.

In the pluripotent stem cells used in the method for evaluating excitotoxicity of the present embodiment, the nucleic acid encoding Ngn2 may be DNA, RNA, or DNA / RNA chimera. Also, the nucleic acid may be double stranded or single stranded. When double stranded, it may be either double stranded DNA, double stranded RNA or a DNA: RNA hybrid. Preferably, it is double stranded DNA or single stranded RNA. For example, when the nucleic acid encoding Ngn2 is a double-stranded DNA (sometimes referred to herein as the Ngn2 gene), the nucleic acid can be introduced into pluripotent stem cells in a form inserted into an appropriate expression vector. . On the other hand, when the nucleic acid encoding Ngn2 is a single-stranded RNA, the RNA may be an RNA into which 5-methylcytidine and pseudouridine have been incorporated in order to suppress degradation, and may be a modified RNA by phosphatase treatment, It is also good. In the present specification, the RNA encoding Ngn2 and the Ngn2 protein may be collectively referred to as the Ngn2 gene product.

In the method of producing nerve cells used in the method for evaluating excitotoxicity according to the present invention, nucleic acid encoding a transcription factor involved in other neurogenesis is pluripotent together with N-factor, as long as the nerve cell induction by N-factor is not inhibited. It may be introduced into sexual stem cells. Such transcription factors include, for example, a nucleic acid encoding Ascl1, a nucleic acid encoding Brn2, a nucleic acid encoding Myt1l, and a nucleic acid encoding HB9.

<Method of introducing nucleic acid>
Although the method of introducing the nucleic acid encoding Ngn2 into pluripotent stem cells is not particularly limited, for example, the following method can be used.

When the nucleic acid is in the form of DNA, it can be introduced into pluripotent stem cells by a method such as lipofection, liposome, microinjection or the like in a form introduced into a vector such as virus, plasmid, artificial chromosome or the like.
Examples of viral vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors and the like. In addition, as a plasmid vector, a mammalian cell plasmid can be used. And as an artificial chromosome vector, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC, PAC) etc. are mentioned, for example. Among these, a plasmid vector and an artificial chromosome vector are preferable, and a plasmid vector is most preferable.

These vectors can contain control sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc., so that the Ngn2 gene can be expressed, and further, if necessary, drug resistance genes (eg, for example) , Kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidine kinase gene, selectable marker sequence such as diphtheria toxin gene, etc., fluorescent protein, reporter gene sequence such as β-glucuronidase (GUS), FLAG etc. .

In another embodiment, the expression cassette (promoter, gene sequence) is inserted into the above-mentioned vector in order to insert the nucleic acid encoding the gene into the chromosome, or to excise the nucleic acid inserted into the chromosome as required. And transposon sequences may be included before and after the gene expression unit containing the Although it does not specifically limit as a transposon sequence, piggyBac is illustrated. In order to introduce an expression cassette into a chromosome using a transposon, it is desirable to introduce transposase into the same cell together with a vector having the expression cassette. In the present invention, in order to introduce transposase, the aforementioned vector may contain the nucleic acid encoding the transposase, and another vector may contain the nucleic acid encoding the transposase, and simultaneously introduced into cells. It is good. Furthermore, the gene product encoding the transposase may be directly introduced. In the present invention, preferred transposases are transposases corresponding to the transposon sequences described above, preferably piggyBac transposases.

When the nerve induction factor is in the form of RNA, it may be introduced into pluripotent stem cells by techniques such as electroporation, lipofection, microinjection, and the like.

When the nerve induction factor is in the form of a protein, it may be introduced into pluripotent stem cells by a technique such as lipofection, fusion with cell membrane permeable peptides (eg, TAT and polyarginine derived from HIV), microinjection and the like.

<Induction of Ngn2 expression>
A nucleic acid encoding Ngn2 can induce expression of Ngn2 at a desired time by functionally joining to an inducible promoter. As such an inducible promoter, mention may be made of a drug responsive promoter, and a preferred example thereof is a tetracycline responsive promoter (a CMV minimal promoter having a tetracycline response element (TRE) having seven consecutive tetO sequences) Can be mentioned. The promoter is a promoter activated by supplying tetracycline or its derivative under expression of reverse tetracycline-regulated transactivator (rtTA; a fusion protein composed of reverse tetR (rTetR) and VP16AD). is there. Therefore, when induction of expression of the gene is performed using a tetracycline responsive promoter, it is more preferable to use a vector having a mode of expressing the activating factor. As a derivative of the tetracycline, doxycycline (doxycycline, hereinafter abbreviated as DOX) can be suitably used.

Moreover, as an expression induction system using a drug responsive promoter other than the above, an expression induction system using an estrogen responsive promoter, a RheoSwitch mammalian inducible expression system using a promoter induced by RSL1 (New England Biolabs) , Q-mate system (Krackeler Scientific) or Cumate inducible expression system (National Research Council (NRC)) using a promoter induced by cumate, and GenoStat inducible expression using a promoter having an ecdysone responsive sequence Systems (Upstate cell signaling solutions) and the like.

When using an expression vector provided with a drug responsive promoter-based expression induction system as described above (ie, a drug responsive induction vector), an agent capable of inducing activation of the promoter (eg, the tetracycline responsive agent) In the case of a vector containing a promoter, expression of Ngn2 can be maintained by continuing to add tetracycline or DOX) to the medium for a desired period of time. Then, expression of the gene can be stopped by removing the drug from the culture medium (for example, replacing it with a culture medium not containing the drug).

Furthermore, expression induction of Ngn2 may be induced by bringing the gene into non-functional form into a constitutive promoter and converting the state of conjugation into a functional state of conjugation at a desired time. As such an example, a specific sequence (for example, a sequence encoding a drug resistance gene or a sequence inducing transcription termination) flanked by LoxP sequences may be inserted between the constitutive promoter and the sequence encoding the gene. A method of converting the bonding state into a functional bonding state, etc., may be mentioned by arranging it and acting Cre at a desired time to remove the sequence sandwiched between the LoxP sequences. Furthermore, instead of the LoxP sequence, FRT sequence or transposon sequence may be used, and FLP (flipase) or the transposon may be used instead of the Cre. In addition, piggyBac transposon is mentioned as a transposon which can be used suitably for this purpose.

Constitutive promoters that can be used for the above purpose include SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine) kinase) promoter, EF-α promoter, CAG promoter and the like.

As described above, when expression is induced by converting the conjugation state using Cre, FLP, and transposon, Cre, FLP, and transposon are allowed to act again after the desired period has elapsed, and the above sequence (LoxP sequence, FRT) The expression of the gene can also be stopped by removing the sequence or the sequence flanked by the transposon sequences).

In another embodiment, the expression period of the gene is controlled by using a vector that can be easily eliminated from the cell, such as an adenovirus vector, an adeno-associated virus vector, a Sendai virus vector, a plasmid, an episomal vector, etc. Is also possible.

In the production of nerve cells, the expression of introduced Ngn2 can exert the effects of the present invention in any of 3 days, 4 days, 5 days, 6 days, and 7 days, and the production of nerve cells is prolonged by becoming long. There is no disadvantage, but it is preferably 3 to 14 days, particularly preferably 7 to 14 days.

<Culture conditions>
In the method for producing nerve cells used in the method for evaluating excitotoxicity according to the present embodiment, a culture medium used to induce differentiation of pluripotent stem cells into which the nucleic acid encoding Ngn2 has been introduced into nerve cells in culture is basically used. A medium alone or a basal medium supplemented with a neurotrophic factor can be used. As such a basic medium, for example, Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) Medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), and a mixed medium thereof are included. The basal medium may contain serum or may be serum free.

As necessary, the medium is, for example, Knockout Serum Replacement (KSR) (serum substitute for FBS in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acid, It may contain one or more serum substitutes such as insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol etc. and also lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential It may also contain one or more substances such as amino acids, vitamins, growth factors, small molecule compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenate, progesterone and putrescine, etc. .

Among them, Neurobasal Medium containing a B27 supplement, or a mixed medium of DMEM and F12 containing insulin, apotransferrin, selenate, progesterone and putrescine can be suitably used as a basic medium.

In the method of producing nerve cells used in the method for evaluating excitotoxicity according to the present embodiment, when differentiation of nerve cells is induced, it can be induced into nerve cells without co-culture with mouse cells, in particular mouse glial cells. Therefore, it is desirable not to carry out co-culture with mouse cells in order to prevent contamination.

In the method for producing nerve cells used in the method for evaluating excitotoxicity according to this embodiment, the culture temperature at the time of induction of differentiation of nerve cells is not particularly limited, and is about 30 to 40 ° C., preferably about 37 ° C. _The culture is carried out under an atmosphere of air containing _{2 and the} concentration of CO ₂ is preferably about 2 to 5%.

Preferably, the exogenous nucleic acid encoding Ngn2 is under the control of an inducible promoter, more preferably under the control of a drug responsive promoter. Pluripotent stem cells into which these foreign nucleic acids have been inserted into the chromosome maintain their undifferentiated ability and high proliferative ability, and can be proliferated while retaining the aforementioned traits. Furthermore, since the above-mentioned trait is not lost even when cryopreservation is performed, it is possible to stably maintain the cell line. In addition, it is a cell capable of rapidly and synchronously differentiating into neural cells by contacting with the agent to which the promoter responds.

<Method for evaluating excitotoxicity to human neurons>
The present invention provides a method for evaluating the excitotoxicity of a test substance to human neurons. The method for evaluating the excitotoxicity of the test substance of the present invention on human neurons comprises the following steps.
(1) bringing a test substance into contact with human neurons produced by inducing differentiation of pluripotent stem cells;
(2) a step of stimulating the human neural cell,
(3) measuring the degree of excitation of the human nerve cell, and (4) if the excitation of the human nerve cell is enhanced in the presence of the test substance, the test substance is excitotoxic to the human nerve cell Process to evaluate that there is

The human neural cells used in the method for evaluating the excitotoxicity of the test substance of the present invention on human neurons may be any human neural cells produced by inducing differentiation of pluripotent stem cells, for example, Examples include human nerve cells produced by inducing differentiation of the pluripotent stem cells described above, and the like. Examples of human neural cells produced by differentiating pluripotent stem cells include cerebral cortical neurons and the like.

The neural cell is preferably a cell that has been passaged once after being induced to differentiate from pluripotent stem cells. As will be described later in the Examples, by passaging once, it is possible to minimize the difference in cell number between wells and the bias in the position of nerve cells in the wells, and to more accurately measure the evaluation of excitotoxicity. It becomes.

For passaging, human neuronal cells are treated with enzymes, isolated and suspended to single cell units, and subjected to cell strainer treatment with a mesh pore size of 40, 70 or 100 μm, preferably 70 μm, and then 5 to 40 × 10 ⁴ Cells / cm ² , preferably at a cell density of 15 × 10 ⁴ cells / cm ² , in Neurobasal medium (manufactured by Thermofisher), B27 supplement without vitamin A (manufactured by Thermofisher) 0.5%, Glutama x 1%, penicillin 100 units It is preferable to carry out by inoculating the culture medium etc. which added / ml and streptomycin 100microg / ml.

Further, it is preferable to inoculate the human neural cells in a culture vessel coated with a coating solution on a culture vessel such as a 96-well clear bottom plate. As the coating solution, 0.5 to 4%, preferably 2% of Matrigel (M coat), 0.002 to 0.005%, preferably 0.003% Poly-L-lysine and 0.5 to 5% are preferable. Mixture (PS coated) with 4%, preferably 1% synthetic human vitronectin substrate (SynthemaxTM II-SC Substrate etc.), 0.002 to 0.005% w / v, preferably 0.003% w / v v Poly-L-lysine and 0.5 to 4%, preferably 1% synthetic human vitronectin substrate (such as SynthemaxTM II-SC Substrate) and 0.5 to 4%, preferably 2% Matrigel Mixture (PSM coat), 0.002-0.005%, preferably 0.003% Poly-L-lysine and 0.5%- %, Preferably 1% synthetic human vitronectin substrate (SynthemaxTM II-SC Substrate etc.) and 0.5% to 4%, preferably 2% Matrigel, 0.002 to 0.005% w / v, Preferably, a mixture (PSMC coat) of 0.003% human type I collagen-like recombinant peptide (Cellnest etc.) and the like are used.

Coat the incubator at room temperature or 37 ° C. for 30 minutes to 4 hours, preferably 2 hours, and then remove the coating solution by suction. The incubator is dried and then seeded with human neurons or immediately seeded with human neurons. Human nerve cells seeded in a culture vessel are subjected to the excitotoxicity evaluation method of the present embodiment 3 to 7 days later.

In the excitotoxicity evaluation method of the present embodiment, the method for stimulating human neural cells is not particularly limited as long as it is a method capable of stimulating human neural cells to cause depolarization. , And methods of adding excitatory nerve synaptic transmitters such as glutamate, and the like.

In the excitotoxicity evaluation method of the present embodiment, the method of measuring the degree of excitation of human nerve cells is not particularly limited as long as it is a method capable of measuring the degree of excitation of nerve cells. Methods of measuring calcium dynamics, methods of measuring changes in fluorescence intensity upon addition of a compound whose fluorescence intensity changes in response to changes in intracellular potential, and the like can be mentioned.

The method of measuring the calcium ion dynamics is not particularly limited as long as intracellular calcium ion dynamics can be measured, and known measurement methods and the like can be used. For example, a method using a calcium indicator dye such as Fluo-8, Fluo-4, fura-2, indo-1 etc., a method using a calcium divalent ion sensitive microelectrode, fluorescence resonance energy transfer (fluorescence) Method using resonance energy transfer (FRET), method using calcium ion-sensitive photoprotein aequorin, calcium sensor protein (for example, green fluorescent protein (EGFP), calmodulin (eg, green fluorescent protein (EGFP)) in which calmodulin protein is genetically linked to a GFP gene variant CaM), a method of expressing and using GCaMP in which myosin light chain fragment (M13) is genetically engineered and the like, and the like can be mentioned. The calcium indicator dye is added to human neurons and, if necessary, washed with PBS, HESS buffer or the like, and then the dye concentration transferred to the cells is measured. Pluronic acid F-127 (manufactured by Sigma-Aldrich) may be used to promote the intracellular transfer of the calcium indicator dye.

Specific examples of the excitotoxicity evaluation method of the present embodiment, in which the degree of excitation of the human neural cells is measured by measuring the intracellular calcium dynamics using electrical stimulation as stimulation to human neural cells, are shown below.

Intracellular calcium dynamics can be measured using FDSS / μCell (manufactured by Hamamatsu Photonics Co., Ltd.). The recording is performed using Excitation 480 nm / Emission 540 nm, and the sampling interval is 30 to 1000 msec, preferably 100 msec. Intracellular calcium dynamics are monitored over time after extracellular field stimulation (EFS) using the stimulation electrodes of the FDSS device. For EFS stimulation electrodes, a 96-electrode array stimulator with an electrical stimulation function is used.

Electrical stimulation (EFS stimulation) of nerve cells is usually performed under conditions of single-phase stimulation, voltage of 20 V, pulse width of 3 msec, and stimulation frequency of 50 Hz. On the other hand, in the excitotoxicity evaluation method of the present embodiment, single-phase stimulation, voltage is 10 to 15 V, preferably 12.5 V, pulse width is 1 to 6 msec, preferably 4 msec, stimulation frequency is 8 to 30 Hz, preferably It is preferable to perform electrical stimulation under conditions of 10 Hz. Thus, by conducting drug studies with weaker extracellular electrical stimulation, it is possible to find minor changes that may be masked by too strong electrical stimulation.

Monitoring is started 30 seconds before EFS stimulation, EFS stimulation is applied with the baseline stabilized before EFS stimulation, and thereafter, intracellular calcium dynamics for about 600 seconds in total is recorded as a change in fluorescence intensity.

The actual waveform of the obtained fluorescence intensity change is monitored over time as ΔF (change intensity) / F0 (baseline intensity) = (100% peak height-bottom height) / bottom height), and ΔF / F0 is the maximum change. The indicated peaks are quantified and the change in ΔF / F0 in the presence of the test substance is measured.

In the method for evaluating excitotoxicity of a test substance of the present invention to human nerve cells, the degree of excitation of the human nerve cell is measured in the presence of the test substance and in the absence of the test substance, respectively. When the degree of excitation of the human nerve cell in the presence of the test substance is enhanced rather than the degree of excitation of the human nerve cell, the test substance can be evaluated as excitotoxic to the human nerve cell .

For example, when the intracellular calcium concentration when electrically stimulated in the presence of a test substance is higher than the intracellular calcium concentration when electrically stimulated in the absence of the test substance, the test substance is a human neural cell. Can be evaluated as excitotoxic.

EXAMPLES The present invention will next be described in more detail by way of examples, which should not be construed as limiting the invention thereto.

Example 1
(Preparation of nerve cells)
A construct expressing human Ngn2 gene under doxycycline control was introduced into induced pluripotent stem cells (iPS cells) established from healthy individuals using a piggyBac vector. It should be noted that depending on the position of genomic insertion of the gene and the amount of introduction, immature neural stem cells positive for NESTIN protein were mixed in addition to mature neurons, and a phenomenon was observed that they gradually replaced neurons when the culture period was extended. Therefore, the seeding density of iPS cells at the time of vector introduction is lowered to 1800 cells / cm ² , and drug resistant selection by G418 is initiated 48 hours after seeding, to obtain single cell-derived colonies of iPS cells into which the construct has been introduced. It was allowed to form and colonies were isolated and established. The established iPS cells were differentiated into about 48 to 120 neural cells, and the appearance thereof was observed, and a strain which was not mixed with weak neural stem cells was selected and used in the following experiment.

The cell lines obtained above were cultured in a medium supplemented with doxycycline for 5 days to induce differentiation into neural cells. Subsequently, differentiation-induced neurons were passaged once. Specifically, differentiation-induced neurons are enzymatically treated with TrypLE (Gibco, manufactured by Themo Fisher Scientific), isolated to single cell units, suspended, and treated with 40 μm mesh pore size cell strainer, 3.3. The cells were seeded at a cell density of 10 ⁵ cells / cm ² . Neurobasal medium is prepared by adding B27 supplement without vitamin A (manufactured by Thermofisher Scientific) 0.5%, Glutamax 1%, penicillin 100 units / ml, streptomycin 100 μg / ml to Neurobasal medium (manufactured by Thermo Fisher Scientific). Using.

The incubator used was a 96-well transparent bottom plate according to SBS standard. The nerve cells prepared above were 0.003% w / v Poly-L-lysine, 1% synthetic human vitronectin substrate (SynthemaxTM II-SC Substrate; Corning), 2% Matrigel and 0.003% The mixture was seeded on a culture vessel coated with a mixture of human type I collagen-like recombinant peptides (manufactured by Cellnest). Coat the incubator at 37 ° C. for 2 hours. The coating solution was aspirated and immediately seeded with cells.

Example 2
(Monitoring intracellular calcium dynamics)
Seven days after cell seeding, 2 μM Fluo-8, an intracellular calcium indicator dye, was added, incubated according to the package insert, and washed with HESS buffer. In order to promote intracellular transfer of the dye, 0.001% w / v Pluronic acid F-127 (manufactured by Sigma Aldrich) was added to the incubation solution.

The N-type calcium channel inhibitor Bepridil (Bourguignon et al. 1989) is adjusted to a maximum concentration of 25 μM, and a 5-fold dilution series is adjusted in seven steps of 1.6, 8, 40, 200 nM, 5, 25 μM. The effect on internal calcium dynamics was evaluated as follows.

FDSS / μCell (manufactured by Hamamatsu Photonics K.K.) was used for measurement of intracellular calcium. For the measurement wavelength, Excitation 480 nm / Emission 540 nm was used, and the sampling interval was 100 msec.
Intracellular calcium dynamics were monitored over time after extracellular field stimulation (EFS) using the stimulation electrodes of the FDSS device. For the EFS stimulation electrodes, a 96-electrode array stimulator with an electrical stimulation function was used.

Electrical stimulation was performed under the conditions of single-phase stimulation, voltage 12.5 V, pulse width 4 msec, and stimulation frequency 10 Hz. Monitoring was started 30 seconds before EFS stimulation, and EFS stimulation was applied with the baseline stabilized before EFS stimulation, and thereafter total intracellular calcium dynamics for 600 seconds was recorded as a change in fluorescence intensity.

In addition, in induction of differentiation of nerve cells, as described above, cell lines are cultured for 5 days, and by differentiating induced differentiation of neurons once, cell number difference between wells can be minimized, and cells by FDSS can be minimized. We were able to minimize the bias that can be caused by the location of neurons in the wells that cause noise in monitoring internal calcium dynamics. This makes it possible to measure intracellular calcium with a weak electrical stimulus with a voltage of 12.5 V, a pulse width of 4 msec, and a stimulation frequency of 10 Hz during electrical stimulation, and may be masked by an electrical stimulus that is too strong A minute change in fluorescence intensity could be found.

DMSO solvent was used as a negative control. The results are shown in FIG. As shown in FIG. 1, in the case of the negative control DMSO solvent, there was no change in ΔF / F 0, but with Bepridil, the change of intracellular calcium was inhibited.

[Example 3]
(Intracellular calcium dynamics upon addition of excitotoxic compounds)
As a test substance, instead of Bepridil, using Amoxapin, Chlorpromazine and Linopirdin, which are agents also used in the United Nations and other research groups on toxicity evaluation systems (HESI), respectively, in the same manner as in Example 2 Calcium kinetics were measured.

The results are shown in FIG. As shown in FIG. 2, with regard to each of Amoxapin, Chlorpromazine and Linopirdin which are known to have neuroexcitotoxicity, Bell-shape type dose-dependent excitotoxicity was confirmed in the excitotoxicity evaluation method of the present invention. It was done.

According to the present invention, it is possible to provide a method for evaluating the excitotoxicity of a test substance to human neurons. The excitotoxicity evaluation method of the present invention can also be applied to a high-throughput screening system in a 96-well format, and can also be applied as a drug drug seed derivation step or as a preclinical in vitro neural excitotoxicity evaluation system for development candidate substances. Useful for therapeutic drug development.

Claims (6)

1. A method for evaluating the excitotoxicity of a test substance to human neurons, comprising
   (1) bringing a test substance into contact with human neurons produced by inducing differentiation of pluripotent stem cells;
   (2) a step of stimulating the human neural cell,
   (3) measuring the degree of excitation of the human nerve cell, and (4) if the excitation of the human nerve cell is enhanced in the presence of the test substance, the test substance is excitotoxic to the human nerve cell A method for evaluating excitotoxicity of a test substance on human neurons, which comprises the step of evaluating the presence of the compound.
2. The method according to claim 1, wherein the human neural cell is a cell that has been passaged once after differentiating pluripotent stem cells into neural cells.
3. The method according to claim 1 or 2, wherein the stimulation to human neural cells is an electrical stimulation.
4. The method according to claim 3, wherein the electrical stimulation is performed at a voltage of 10 to 15 V and a stimulation frequency of 8 to 30 Hz.
5. The method according to any one of claims 1 to 4, wherein the step of measuring the degree of excitation of human neurons is the measurement of calcium dynamics in the human neurons.
6. The method according to any one of claims 1 to 5, wherein the human neuronal cell is a cerebral cortical neuronal cell.

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