JP5783504B2 - Methods for assessing impact on embryonic programming - Google Patents

Description

  The present invention relates to a method for creating a neuronal cell model, a neurosphere precursor composed of cells derived from ES cells used therein, a method for measuring the effect on embryonic programming using the neuronal cell model, and the effect on embryonic programming The present invention relates to a method for evaluating the risk, a method for predicting the risk of lesions, and a method for screening substances having an effect on embryonic programming.

  The situation surrounding the biological effects of chemical substances includes lack of information on chemical substances, an increase in health effects that have been overlooked in traditional risk assessments such as allergic diseases and nervous system disorders, and further, these effects are caused by exposure to prenatal exposure. There are some indications that this is a developmental effect (embryonic programming). It is necessary to clear these indications when managing biological risks (biological health risks). For this purpose, there is a demand for the construction of a toxicity evaluation system that can evaluate not only a wide variety of chemical substances but also a wide variety of endpoints, and that can analyze simple and high-speed specimens. Furthermore, establishment of a model cell line that mimics the development and developmental stages of adult organs is also desired.

  A method using neuroblasts has also been reported as a method for evaluating the influence on neurological dysfunction (Patent Document 1), but a method for evaluating embryonic programming has not yet been developed.

JP 2006-325551 A

  The present invention provides a fetus programming evaluation system that can easily and rapidly analyze a multi-specimen.

  As a result of intensive studies in order to solve the above-mentioned problems, the present inventors use a neurosphere system model, a neurosphere precursor composed of cells derived from ES cells used therefor, and the above-described neuronal cell model. Developed a method for measuring the effect on embryonic programming, a method for assessing the effect on embryonic programming, a method for predicting lesion risk and a method for screening substances having an effect on embryonic programming, Completed the invention.

That is, the present invention is as follows.
[1] A neurosphere precursor composed of cells derived from ES cells for creating a neuronal cell model.
[2] The neurosphere precursor according to [1], wherein the ES cell is a human ES cell.
[3] The following steps:
Exposing microspheres composed of ES cells to nerve inducers to form neurosphere precursors;
A method for creating a neuronal cell model, comprising differentiating cells constituting neurospheres into mature neurons by culturing the obtained neurosphere precursor in a nerve induction medium using a container for neuronal cell culture .
[4] The method for creating a nerve cell system model according to [3], wherein the nerve cell culture container is an ornithine / laminin coated plate.
[5] The method for creating a neuronal cell model according to [4], wherein all steps are completed in 20 days.
[6] The following steps:
Contacting a sample with a microsphere composed of ES cells;
Separating a sample from the microsphere;
Exposing a part of the microsphere to a nerve inducer to form a neurosphere precursor, collecting total RNA from the remaining part, and measuring the expression level of the gene;
Creating a neuronal cell system model for differentiating the neurosphere-forming cells into mature neurons by culturing the obtained neurosphere precursor in a nerve induction medium using a neuron culture vessel,
A method for measuring the effect on embryonic programming comprising measuring the cell morphology of the resulting neural cell lineage model.
[7] The method for measuring an influence on embryonic programming according to [6], wherein the nerve cell culture container is an ornithine / laminin coated plate.
[8] The gene is as follows:
One or more nuclear receptor genes selected from the group consisting of ERα, ERβ, AR, RARα and RARβ;
One or more susceptibility genes for schizophrenia selected from the group consisting of DTNBP1, NRG1, DAO, DAOA, RGS4, CAPON, PPP3CC, TRAR4, VCFS, COMT, PRODH, DHHC, ZDHHC8, DISC1 and GRM5,
One or more autism-related genes selected from the group consisting of Tsc1, Tsc2, Fmr1, Ube3a, Reln, Nlg3, Foxp2, Cntnap2, Slc6a4, Gabrb3, Mecp2 and En2,
One or more axon guidance-related genes selected from the group consisting of EphrinB, EphB, Sema3A, PlexinA, Sema7A, Itgb1, Netrin1, Slit1, Robo2, Cxcl12, Cxcr4, NetrinG, NGL1, Unc5,
One or more brain segmentation-related genes selected from the group consisting of Foxg1, Emx1, Emx2, Nkx2.1, Otx1, Otx2, En1, Gbx2, Hoxb1 and Hoxa2;
Snca, Uchl1, Apoe, Park7, Apbb1, Bcl2, Bcl2, Ube2l1, Ubqln1, Bax, Cdk5, Ubb, Als2, Gtf2a1, a group consisting of Base1, App, Psen1, Ide, Ccs, and Sod1Gp, and Sod1Gp, Sod1Gp Disease-related genes,
Nanog, Klf4, Zfp42, Pou5f1, Fgfr1, Nestin, Tuj1, Map2, Olig2, Gfap, Raf1, Atbf1, Pla2g6, Cdyl, Mapk3, Shc1, Hras1, Rps6k1, Hps1, Rps6k One or more neurodevelopmental genes selected from the group consisting of:
One or more Parkinson's disease-related genes selected from the group consisting of Th, Slc6a3, Snca, Ube1, Ubch7, Park2, Uch1, Park7, Casp9, Casp3 and Casp7, and / or App, Bace, Psen, ApoE, Ide, Mme A method for measuring the effect on embryonic programming according to [7], which is a combination with one or more Alzheimer's disease-related genes selected from the group consisting of Il1r1, Tnfrsf1a, Casp3 and Casp7.
[9] The cell morphology is neurosphere area, neurosphere roundness, number of neurospheres, number of cells, nucleus area, neurosphere circumference, neurite length, number of neurite intersections And the number of neurite branching points according to [7], the method for measuring the influence on embryonic programming.
[10] The method for measuring an influence on embryonic programming according to [9], wherein the measurement of the cell morphology is performed by a multichannel cell image analyzer.
[11] The measured values of the gene expression level and the cell morphology obtained by the method according to [6] are compared with the measured values for substances not treated with a sample or known to have an effect on embryonic programming. To evaluate the impact on embryonic programming.
[12] The method for evaluating the influence on the embryonic programming according to [11], wherein the influence on the embryonic programming is evaluated using a Bezier network system.
[13] A lesion risk comprising comparing the measured value of the gene expression level and the cell morphology obtained by the method according to [6] with a measured value of a substance having a known effect on embryonic programming. Method to predict.
[14] In order to screen for a substance having an influence on embryonic programming, in which the measured value of the gene expression and the measured value of the cell morphology obtained by the method according to [6] are compared with the measured value when not treated with a sample. the method of.
[15] The method for screening according to [14], wherein the substance having an influence on embryonic programming is an environmental chemical substance, a pharmaceutical product, or an agricultural chemical.

  By using the method of the present invention, the impact on embryonic programming can be evaluated. In addition, if a high-throughput analysis of late effects due to fetal exposure is performed using the method of the present invention, it is possible to use information for predicting biological (health) effects on a wide variety of chemical substances. This makes it possible to predict the impact on the living body (health) and to prevent damage from exposure to chemical substances.

FIG. 2 is a schematic diagram of the method of the present invention. It is a network figure of the multi information of a control group (0.1% DMSO). It is a network figure of the multi information of a PCB (10 ^<-8> M) treatment group. It is a network figure of the multi information of an E2 (10 ^<-8> M) treatment group. It is a network figure of the multi information of a PMT (10 ^<-5> M) treatment group. It is a network figure of the multi information of a DHT (10 ^<-8> M) treatment group. It is a network diagram of the multi information of a TMD (10 ^<-5> M) treatment group. It is a network diagram of multi information of a BPA (10 ⁻¹⁰ M) treatment group. It is a network figure of the multi information of a DEHP (10 ^<-4> M) treatment group. It is a network figure of the multi information of a T3 (10 ^<-8> M) treatment group. It is a network diagram of multiple information of MPA (10 ⁻⁴ M) treatment group. It is a network diagram of the multi information of a DEX (10 ^<-8> M) treatment group. It is a network figure of the multi information of a CPM (10 ^<-5> M) treatment group. It is a multi-information network diagram of the TCDD (10 ⁻⁸ M) treatment group.

  In the present invention, a neuronal cell model is an area ratio occupied by neurons (MAP2-positive area) of about 20 to 50% of the total cell area, and an area ratio occupied by glial cells (area occupied by GFAP-positive cells). It is about 50 to 80% of the total cell area.

  In the present invention, an ES cell refers to a cell that is made from an inner cell mass belonging to a part of an embryo in the blastocyst stage, which is an early stage of animal development, and has pluripotency and infinite proliferation ability. The animal is preferably a mammal, more preferably a mouse or a human, and most preferably a human. In the present invention, iPS cells can also be used instead of ES cells.

  In the present invention, the neurosphere precursor refers to a cell aggregate that expresses a neural stem cell marker or an early neurogenesis marker but does not generate a glial cell. For the differentiation of mature neurons, the average diameter of the neurosphere precursor is preferably 640 μm.

  In the present invention, the microsphere refers to a spherical mass in which 45 to 200 cells, preferably 50 to 150 cells, more preferably 98 cells are aggregated. For the differentiation of mature neurons, the average diameter of the neurosphere precursor is 210 μm (the diameter just before seeding on the ortinin laminin coat is 210 μm, and on the 20th day after culturing in the ortinin laminin coat, the average diameter of the neurosphere is 640 μm). The average diameter of the microspheres is preferably 100 to 300 μm, more preferably 200 to 250 μm, and most preferably 210 μm.

In the present invention, the nerve inducer refers to a substance having the ability to induce undifferentiated cells into nerve cells, and examples thereof include retinoic acid. The treatment concentration of retinoic acid is preferably high (10 ⁻⁶ M), medium (10 ⁻⁷ M) or very low (10 ⁻⁹ M), more preferably low (10 ⁻⁸ M). M).

  In the present invention, the nerve cell culture vessel refers to a culture instrument for culturing ES cells before nerve induction to make the size and differentiation speed uniform and to form a neurosphere precursor, for example, a microdevice. . Preferably, it is a 10-60 mm dish, More preferably, it is a 24-well plate, Most preferably, it is a three-dimensional patterning (or microsphere array).

  The nerve cell culture container of the present invention is preferably coated with ornithine / laminin. An ornithine / laminin coated plate can be prepared by adding an ornithine solution to the plate, allowing it to stand at 37 ° C. overnight, washing it with water, adding a laminin solution, and allowing it to stand at 37 ° C. for 4 hours or longer. BD Bio Coat ™ poly-L-Ornithine / Laminin cell ware 24-well plates are commercially available as ornithine / laminin coated plates. However, this plate must be used with ornithine / laminin coated on its own because of the poor adhesion of neurosphere precursors and poor differentiation induction and proliferation into mature neural stem cells.

In the present invention, a mature nerve cell refers to a cell that satisfies all of the following conditions.
1) MAP2 having 1000 to 3500 bifurcation points per one visual field area (33335547 μm ² ) of neurons, 200 to 600 intersections per visual field area, and (33335547 μm ² ) per visual field area The total neurite extension (total extension) of positive neurons is 10,000 μm to 500,000 μm;
2) GFAP-positive glia that have 150 to 2700 branch points per visual field area, 20 to 500 intersections per visual field area, and (33335547 μm ² ) per visual field area The total elongation (total elongation) of cytoplasmic processes of the cells is 90000 μm to 300000 μm,
3) Neurons and glial cells are derived from neurosphere precursors consisting of 10,000 to 50,000 cells, and these cells have migrated and spread,
4) MAP2 positive total area is 10000μm ² ~2000000μm ^2, GFAP-positive area in the range of 600μm ² ~4000000μm ^2.

In the present invention, total RNA can be recovered using any conventionally known method. For example, Total RNA can be purified from a neurosphere precursor 48 hours after chemical exposure on a microsphere array according to the technique of RNeasy Mini Kit (50) (QIAGEN). In detail, neurosphere precursors cultured on a microsphere array were washed with PBS, collected by pipetting, centrifuged at 1000 rpm for 5 minutes, PBS was removed, and 350 μl per 5 × 10 ⁶ ES cells. Of RLT Buffer is added and removed 10 times with a 1 ml syringe 23G (diameter 0.65 mm) to disperse the cells. The cells are stored at −80 ° C. until subjected to testing. Add 350 μl of 70% ethanol and pipette until cloudy. Further, 700 μl of the sample is placed in an RNeasy spin column placed on a 2 ml collection tube. Next, centrifuge at 12000 rpm for 1 minute at room temperature. Discard the solution in the lower column. Further, 700 μl of RW1 buffer contained in the RNeasy Mini Kit (50) (QIAGEN) kit is added, and the mixture is centrifuged at 12000 rpm at room temperature for 1 minute. Next, 80 μl of DNase I solution is added and left at room temperature for 15 minutes. Next, 350 μl of RW1 is added and centrifuged at 12000 rpm for 1 minute at room temperature, 500 μl of RPE buffer is added and centrifuged at 12000 rpm for 1 minute at room temperature. Add 500 μl RPE buffer, centrifuge at 12000 rpm for 1 min at room temperature, centrifuge at 15000 rpm for 1 min at room temperature and transfer the spin column to a new 1.5 ml collection tube. Next, 20 μl of RNase-free water is added and centrifuged at 10,000 rpm at room temperature for 1 minute to obtain total RNA.

  In the present invention, the expression level of a gene can be measured using any conventionally known method. For example, gene expression levels can be analyzed by Illumina Bead Array. After measuring the absorbance of the extracted total RNA, an electrophoretic analysis for a total amount of the total RNA was performed using Agilent 2100 bioanalyzer and RNA LabChip Kit. In order to perform the Illumina Bead Array, the sample amount must be 500 ng / 10 μl or more per array, and the sample purity should be 0.1.260 / 0.1.280 = 1.7 to 2.1. is there.

  In the present invention, cell morphology refers to the area of neurosphere, the roundness of neurosphere, the number of neurospheres, the number of cells, the area of nucleus, the circumference of neurosphere, the length of neurites, the number of intersections of neurites And the number of branch points of the neurite. This cell morphology can be measured using a multichannel cell image analyzer.

  The multi-channel image analyzer that can be used in the present invention is, for example, IN Cell Analyzer 1000 (GE Healthcare Bioscience), and the cell morphometry method is morphological analysis with an image analyzer after culturing for 20 days. For this purpose, fixation was performed with 4% PFA (paraformaldehyde) -0.2 M sucrose-0.1 M PBS (pH 7.4) for about 15 minutes. After fixation, the plate was washed three times with 0.1 M PBS, and then treated with 0.1% Triton X 100-0.1 M PBS for 30 minutes as a permeation treatment. Thereafter, the plate was washed 3 times with 0.1 M PBS and subjected to a blocking operation with 1% BSA (bovine serum albumin) -0.1 M PBS for 10 minutes.

  Mouse anti-nestin monoclonal antibody (CHEMICON International), which is a specific marker for neural stem cells, and mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (CHEMICON International), which is a marker for glial cells, are selected as primary antibodies for use in the present invention. And allowed to react overnight at 4 ° C. The next day, the cells were washed 3 times with 0.1M PBS, reacted with a labeled goat anti-mouse IgG antibody (Alexa Fluor 568, Invitrogen) as a secondary antibody for 1 hour at room temperature, and then 3 times in 0.1M PBS for 10 minutes. Wash one by one. As a nuclear stain, after reacting with 2 μg / ml Hoechst 33342 solution (Dojindo) for 15 minutes, the cells are washed 3 times with 0.1 M PBS for 5 minutes. In order to perform analysis with the IN Cell Analyzer 1000, each well is filled with PBS to about 80%, covered with parafilm to prevent drying, sealed with a dish lid, and stored in a refrigerator. After being acquired as an image by a multi-channel cell image analyzer (IN Cell analyzer 1000, GE), the area of the neurosphere, the roundness of the neurosphere, the number of neurospheres, the number of cells, the area of the nucleus, the circumference of the neurosphere, The length of neurites, the number of neurite intersections and the number of neurite branching points are selected as parameters, and the morphology of individual neurons is automatically measured by the Developer Tool Box, one of the multi-analysis software. Conversion to information, and comparative analysis of the effects of chemical substances on neuronal differentiation as quantitative and objective phenotype data.

The genes that can be used in the present invention are as follows:
Raf1 (GENBANK ID AK036317), Atbf1 (NM_007496), Pla2g6 (NM_016915), Cdyl (NM_009881), Mapk3 (NM_011952), Shc1 (NM_011368), Hras1 (NM_0082k1, NM_0082k) ), Gbx2 (NM — 010262), Sall1 (NM — 021390), Map2k1 (NM — 008927), Fos (NM — 010234), Rif1 (NM — 175238), Gfap (NM — 010277), Map2 (NM — 008927), Nestin (M 1 or more and neuronal differentiation genes selected,
Esr1 (AK054182), Esr2 (NM_010157), AR (NM_013476), RARa (NM_031528), RARb (Mm.259318), RARg (NM_011244), Nanog (NM_0280361), Klf4 (Z_f053636P, ), Fgfr1 (NM — 010206), Nestin (NM — 016701), Tuj1 (NM — 023279), Map2 (NM — 008927), Olig2 (NM — 016967), Gfap (NM — 010277), and one or more nuclear receptor genes selected from the group A marker gene,
Esr1 (AK054182), Esr2 (NM_010157), AR (NM_013476), RARa (NM_031528), RARb (Mm.259318), RARg (NM_01120244), EphrinB (NW_001030882), EphB (NM_173mA, SNP47Min) .3789), Sema7A (NM_011352), Itgb1 (NM_010578), Netrin1 (Mm.39095), Slit1 (NM_015748), Robo2 (NM_175549), Cxcl12 (NM_013655), Mxcr99 (G00N1) Mm.241682), Unc5 (NM_153) And axonal extension comprising one or more of the nuclear receptor gene selected from the group consisting of 31),
Esr1 (AK054182), Esr2 (NM_010157), AR (NM_013476), RARa (NM_031528), RARb (Mm.259318), RARg (NM_011244), Tsc1 (NM_022878), Tsc2 (NM_011647) N_011647N_011647N_011647 ), Reln (NM — 012661), Nlgn3 (NM — 172932), Foxp2 (NM — 053242), Cntnap2 (NM — 001004357), Slc6a4 (NM — 010484), Gabrb3 (NM — 008071), M012 from M01107 Autism-related genes including nuclear receptor genes of
Esr1 (AK054182), Esr2 (NM_010157), AR (NM_013476), RARa (NM_031528), RARb (Mm.259318), RARg (NM_011244), Th (NM_009377), Slc6a3 (NM_010020), Nc010020N ), Ubch7 (NM_02069), Park2 (NM_016694), Uchl1 (NM_011670), Park7 (NM_02069), Casp9 (NM_015733), Casp3 (NM_009810), and Casp7 (NM_007611). Parkinson's disease related genes including genes,
Esr1 (AK054182), Esr2 (NM_010157), AR (NM_013476), RARa (NM_031528), RARb (Mm.259318), RARg (NM_011244), App (NM_007471), Base (NM_011712), M ), Ide (NM_031156), Mme (NM_008604), Il1r1 (NM_008362), Tnfrsf1a (NM_011609), Casp3 (NM_009810), Casp7 (NM_007611), and including Alzheimer's disease Combination with related genes.

  In the present invention, a method for comparing the measured value of the gene expression level and the above cell morphology with the measured value for a substance that is not treated with a sample or has a known effect on embryonic programming is, for example, a Vegian network system. is there. A Bayesian network is a kind of probabilistic model that can be used for predicting events including uncertainty, rational decision making, fault diagnosis, and the like. A certain probability distribution is expressed, the probability distribution is calculated, and prediction and optimum decision making are performed. This calculation of the probability distribution is called probability inference. A characteristic of Bayesian networks is that a causal structure is represented as a network, and probabilistic reasoning is performed on the network to predict the likelihood and possibility of uncertain events. For example, if the relationship that “the variable (parameter) X takes the value x (parent node), the variable Y becomes y (child node)” is established, the value taken by Y is independent of the value of X. is not. That is, it can be said that Y depends on the value of X and has a covariant relationship. TAOgen uses linear regression to calculate the relationship between acyclic networks developed by Toyoshiba et al. And Yamanaka et al., And clarify linkages between genes (Yamanaka T, Toyoshiba H , Sone H, Parham FM, Portier CJ .: “TAO-Gen algorithm for distinguishing gene interactions and networks by application to SOS repair in E. coli” Environ Health Perspect. 112, 16, 1614-1621 (2004 ), Toyoshiba H, Yamanaka T, Sone H, Parham FM, Walker NJ, Martinez J, Portier CJ .: “Gene interactions and networks are important links between dioxins aryl hydrocarbon receptors and retinoic acid receptors beta. "Environ Health Perspect. Vol. 112, No. 12, pp. 1217-1224 (2004).). Based on this TAOgen, analysis was performed using a software called multi-TAOgen, which was uniquely customized.

  In the present invention, to compare the measured value of the gene expression level and the above-mentioned cell morphology with the measured value of a substance having a known influence on embryonic programming means to construct an integrated network by multi-TAOgen software. .

  In order to evaluate late effects after fetal exposure, uniform embryoid body (EB) from ES cells on an array with 100-1020 microparticle-shaped grooves capable of uniformly controlling the size of ES cells. After that, a culture method was established for inducing differentiation into mature neurons by culturing on the nerve-derived basement membrane.

For example, with a retinoic acid (10 ⁻⁸ M) that is a nervous system inducer, exposure to a chemical substance for a certain period of time (eg, 48 hours) from the start of culture on a three-dimensional patterning array, followed by RNA extraction and gene expression Measure. Furthermore, it culture | cultivates on a nerve induction | guidance | derivation basement membrane, and the form of a mature nerve cell is measured. The measured gene expression information and cell morphology information are shown in a network diagram that shows the interrelationships stochastically by the Bayesian algorithm, and the difference in influence is shown by the difference in network structure. These series of methods are the method of detecting the influence of the present invention on the embryonic programming of chemical substances by multi-profiling.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not limited to this.

1. Preparation of ES cell B6G-2 cell line (RIKEN), a mouse embryonic stem cell line (mES cell) derived from the green mouse FM131 mouse prepared by linking the CAG promoter to the GFP expression vector, was subjected to growth inactivation treatment. Fully grown on fibroblasts (MEF). Thereafter, on a 60 mm dish coated with 0.1% gelatin, DMEM (without phenol red, Invitrogen), 15% FBS (fetal calf serum, Invitrogen), 100 μM NEAA (Non-essential amino acids, Invitrogen), 100 μM 2- Subculture was performed in ES medium containing ME (2-mercaptoethanol, Invitrogen) and 1000 U / ml LIF (leukemia inhibitory factor, ESGRO, Invitrogen). After the second passage in which the influence of MEF was minimal and gene mutation was suppressed, ES cells were seeded on the microsphere array.

2. Preparation of ES cell microspheres Controlling the environment in which cells are cultured is extremely important in understanding cell behavior and applying cell functions in engineering. In this research, a microsphere array of a device that has been subjected to a microfabrication technology (soft lithography) that patterns the substrate to which cells adhere, the position and shape of the attachment surface, and the flow field around the cells with micrometer precision. Above, the size and differentiation rate of mouse ES cells were homogenized to establish a three-dimensional culture method for inducing mature neurons. The structure of the frame-separated microsphere array (chip 300) used in this study is made of acrylic resin as the substrate, the outer dimensions are 24 x 24 mm square, and the frame resin is made of PDMS resin. The shape is designed as a frame shape with a volume of 250 μl of medium inside. This frame can be easily removed with tweezers. Further, 1020 uniform wells are formed in the frame (10 × 10 mm), the well diameter is 300 μm, the distance (pitch) between the wells is 330 μm, and the well depth is 270 μm. is there.

This frame-separated microsphere array (chip 300) was transferred to a 6-well plate, and immediately after removing all the sterilized water in the frame with a pipetman, the EB medium was added and replaced with PBS (−). After performing this operation three times, 250 μl of EB medium was added and left in an incubator at 37 ° C. The ES cell suspension at the second passage was adjusted to 4 × 10 ⁵ cells / ml. After adjustment, the microsphere array in the incubator is taken out, the EB medium is removed by aspiration, and the ES cell suspension is slowly poured into the array frame at 1 × 10 ⁵ cells / 250 μl from the end of the frame for 4 hours in the incubator. I left it still. It was confirmed that all ES cells seeded in the array were uniformly accumulated in the well within 10 minutes. After culturing for 6 hours, the medium was replaced with an EB medium added with a chemical substance, and the chemical substance was exposed for 48 hours.

3. Preparation of Neurosphere Precursor 48 hours after addition of chemical substances, the medium was replaced with an EB medium containing 10 ⁻⁸ M retinoic acid. On the fourth day of the culture, the medium was replaced again with an EB medium containing 10 ⁻⁸ M retinoic acid. Furthermore, on the 6th day of culture, the medium was replaced with EB medium only. On the 8th day of culture, the microsphere array was removed with tweezers, and lightly pipetted with a 1 ml pipette in a non-adhesive 35 mm dish to completely recover all neurosphere precursors. Collected in a 5 ml microtube. The collected neurosphere precursors were seeded on a 24-well ortinin / laminin-coated plate made in advance with a 200-micron chip so that the collected neurosphere precursor was uniformly 83 / well with a 200-micron chip. The average diameter of the neurosphere precursor at this time was about 210 μm.

  The production method of ortinin / laminin coat is as follows. The Poly-1-ornithine solution was diluted 2.5 times with distilled water. 100 μl laminin was diluted with 12 ml PBS. A 2.5-fold diluted Poly-1-ornithine solution was dispensed at 500 μl into 24 wells and allowed to stand overnight in a 37 ° C. incubator. The next day, it was washed 3 times with DW. 500 μl of diluted laminin was dispensed into 24 wells and allowed to stand in a 37 ° C. incubator for 4-6 hours. Laminin was completely aspirated with an aspirator, 1 ml of EB medium was added, and the mixture was allowed to stand in a 37 ° C. incubator.

4). Preparation of a neuronal cell line model After culturing in an EB medium for 24 hours, the neurosphere precursor was completely adhered in the well, and after confirming short neurite outgrowth, a nerve induction medium (DMEM / F12 (1: 1), N2 The medium was changed to 10 μg / ml bFGF). The medium was changed every 3 days. After seeding the neurosphere precursor on the ortinin laminin coat, the culture was carried out for 12 days (the total culture period was 20 days). Cells on the 12th day of culture (20 days in total for the culture period) met all of the following conditions.
(1) The average roundness of the neurosphere precursor is 0.49, (2) The average circumference of the neurosphere precursor is 1551.7 μm, (3) The average total area of the neurosphere precursor is 6654069.2 μm ² (4) The average number of neurosphere precursors is 84.7, (5) The average diameter of neurosphere precursors is 492.3 μm, and (5) The total number of cells migrating from the neurosphere precursor is 14468. 4. (6) The average total area of MAP2-positive neurosphere precursors is 601939.8 μm ² , (7) The average total extension of neurites of MAP2-positive neurons migrating from neurosphere precursors is 237214.1 μm, (8) The average total number of intersections of MAP2-positive neurons is 1284.6, (9) The average total number of branch points of MAP2-positive neurons is 4615.6, ( O) Mean total area of GFAP-positive neurospheres precursor is, 719544.9μm ^2, (11) average total stretch of cytoplasmic GFAP-positive cells migrating from neurospheres precursor is, 43151.3μm (12) GFAP positive cells The average total number of crossing points was 227.4, and the average total number of branch points of (12) GFAP positive cells was 1079.5.

5. Results of Experiments with 12 Chemical Substances Using the IN Cell Analyzer 1000 of the multi-channel image analyzer, the control group and 12 kinds of selected test chemicals (10 nM triiodothyroxine, 10 nM dexamethasone, 10 nM 17β as high doses, respectively) -Estradiol, 10 nM 5α-dihydrotestosterone, 10 nM 2,3,7,8-tetrachlorodibenzo-para-dioxin (tetrachlorodibenzodioxin), 100 μM methotreic acid, 10 μM cyclopamine, 10 μM thalidomide, 10 nM 4 (OH) -2 ′, 3,3 ′, 4 ′, 5′-pentachlorobiphenyl (polychlorobiphenyl hydroxide 107), 10 μM permethrin, 100 pM bisphenol-A, 100 μM bis (2-ethylhexyl) Le) shows the result of exposure effect of phthalic acid (diethylhexyl phthalate) below.

(1) The roundness of the neurosphere precursor is 0.49 for the control group, 0.54 for 10 nM Dex, 0.56 for 10 μM PMT, 0.56 for 10 nM E2, 0.50 for 10 nM TCDD, 10 nM DHT is 0.53, 100 μM DEHP is 0.57, 100 pM BPA is 0.58, 10 nM PCB is 0.51, 10 nMT3 is 0.52, 10 μM TMD is 0.51, 10 μM CPM is 0 .52, 100 μM MPA was 0.55.

(2) The circumference of the neurosphere precursor is 1551.7 μm for the control group, 1436.7 μm for 10 nM Dex, 10 μM for PMT is 1187.8 μm, 10 nM E2 is 1250.5 μm, 10 nM TCDD is 1429.9 μm, 10 nM DHT is 1393.0 μm, 100 μM DEHP is 1207.8 μm, 100 pM BPA is 1182.6 μm, 10 nM PCB is 1565.8 μm, 10nMT3 is 1362.5 μm, 10 μM TMD is 1317.0 μm, 10 μM CPM is 1353.6 μm, 100 μM MPA was 1269.2 μm.

(3) the area of the neurosphere precursor is control group 6654069.2μm ^2, 10nM Dex is 6746902.5μm ^2, 10μM PMT is 5540044.2μm ^2, 10nM E2 is 4909425.8μm ^2, 10nM TCDD is 5446502.0Myuemu ² , 10nM DHT is 6109497.7μm ^2, 100μM DEHP is 5031102.7μm ^2, 100pM BPA is 2756341.9μm ^2, 10nM PCB is, 4488292.4μm ^2, 10nMT3 is, 6088273.9μm ^2, 10μM TMD is 4142275.1μm ^2, The 10 μM CPM was 41644921.6 μm ² and the 100 μM MPA was 4197825.1 μm ² .

(4) The number of neurosphere precursors is 84.7 in the control group, 83.2 in 10 nM Dex, 89.6 in 10 μM PMT, 69.5 in 10 nM E2, and 72.9 in 10 nM TCDD. 10 nM DHT is 76.3, 100 μM DEHP is 73.6, 100 pM BPA is 42.3, 10 nM PCB is 50.1, 10 nMT3 is 81.7, 10 μM TMD is 59.2, There were 54.6 10 μM CPM and 58.8 100 μM MPA.

(5) The diameter of the neurosphere precursor is 492.3 μm for the control group, 459.4 μm for 10 nM Dex, 10 μM PMT for 374.8 μm, 10 nM E2 for 400.9 m, 10 nM TCDD for 455.8 μm, 10 nM DHT Is 447.8 μm, 100 μM DEHP is 383.9 μm, 100 pM BPA is 365.0 μm, 10 nM PCB is 501.3 μm, 10 nMT3 is 431.8 μm, 10 μM TMD is 418.1 μm, 10 μM CPM is 429.6 μm, 100 μM MPA Was 402.4 μm.

(5) The number of cells migrating from the neurosphere precursor is 14468.4 in the control group, 35288.1 in the 10 nM Dex, 33720 in the 10 μM PMT, 28912.1 in the 10 nM E2, and 13155 in the 10 nM TCDD. 1, 10 nM DHT 32571.9, 100 μM DEHP 33674.4, 100 pM BPA 10271.4, 10 nM PCB 10319.8, 10 nMT3 32269.9, 10 μM TMD 22190.1 10μM CPM was 20452.1 and 100μM MPA was 26177.6.

(6) area of MAP2-positive neurospheres precursor is control group 601939.8μm ^2, 10nM Dex is 1077594.6μm ^2, 10μM PMT is 1049239.8μm ^2, 10nM E2 is 732158.4μm ^2, 10nM TCDD is 471,940. 7μm ^2, 10nM DHT is 254330.6μm ^2, 100μM DEHP is 570495.2μm ^2, 100pM BPA is 344525.8μm ^2, 10nM PCB is, 354876.3μm ^2, 10nMT3 is, 96074.0μm ^2, 10μM TMD is 76430.1μm ² , 10 μM CPM was 100555.5 μm ² , and 100 μM MPA was 198445.2 μm ² .

(7) The total extension of neurites of MAP2-positive neurons migrating from neurosphere precursors was 2372.14.1 μm for the control group, 380272.0 μm for 10 nM Dex, 10293344.0 μm for 10 μM PMT, 250986.2 μm for 10 nM E2. 10 nM TCDD is 183235.9 μm, 10 nM DHT is 1414267.0 μm, 100 μM DEHP is 224278.9 μm, 100 pM BPA is 125299.8 μm, 10 nM PCB is 137283.3 μm, 10nMT3 is 80916.1 μm, 10 μM 1017.5 m The 10 μM CPM was 26316.2 μm and the 100 μM MPA was 42969.9 μm.

(8) The number of intersections of MAP2-positive neurons is 1284.6 for the control group, 1404.3 for 10 nM Dex, 1482.5 for 10 μM PMT, 955.6 for 10 nM E2, and 987.4 for 10 nM TCDD. 10nM DHT is 543.8, 100μM DEHP is 869.2, 100pM BPA is 664, 10nM PCB is 725.8, 10nMT3 is 303.4, 10μM TMD is 130.4, 10μM CPM was 141.4 and 100 μM MPA was 245.2.

(9) The number of branch points of MAP2-positive neurons is 4615.6 in the control group, 6548.9 in 10 nM Dex, 6230.7 in 10 μM PMT, 43272.5 in 10 nM E2, and 3525 in 10 nM TCDD. 6, 2157.3 for 10 nM DHT, 3630.8 for 100 μM DEHP, 2395.6 for 100 pM BPA, 2573.7 for 10 nM PCB, 1127 for 10 nMT3, 448.1 for 10 μM TMD, The number of 10 μM CPM was 481.4, and the number of 100 μM MPA was 806.6.

(10) the area of the GFAP-positive neurospheres precursor is control group 719544.9μm ^2, 10nM Dex is 2029240μm ^2, 10μM PMT is 955109.9μm ^2, 10nM E2 is 831347.6μm ^2, 10nM TCDD is 569219.1Myuemu ² , 10nM DHT is 1808531.8μm ^2, 100μM DEHP is 1086888.7μm ^2, 100pM BPA is 1100168.1μm ^2, 10nM PCB is, 1129661.4m ^2, 10nMT3 is, 207471.2μm ^2, 10μM TMD is 213093.5μm ^2, The 10 μM CPM was 109251.6 μm ² and the 100 μM MPA was 252321.3 μm ² .

(11) The total elongation of cytoplasmic processes of GFAP positive cells migrating from neurosphere precursors is 43151.3 μm in the control group, 125382.9 μm in 10 nM Dex, 1038 μm in PMT, 43876.0 μm in 10 nM E2, 38771.9 μm in E2 10 nM TCDD is 34419.4 μm, 10 nM DHT is 19023.13.1 μm, 100 μM DEHP is 57664.5 μm, 100 pM BPA is 61513.8 μm, 10 nM PCB is 65410.1 μm, 10nMT3 is 40538.4 μm, 10 μM TMD is 82679.9 μm. The 10 μM CPM was 57194.8 μm, and the 100 μM MPA was 49229.4 μm.

(12) The number of intersections of GFAP positive cells is 227.4 in the control group, 407.9 in 10 nM Dex, 136 in 10 μM PMT, 116.1 in 10 nM E2, 184.3 in 10 nM TCDD, 10 nM DHT is 404.7, 100 μM DEHP is 169.1, 100 pM BPA is 302.7, 10 nM PCB is 322.6, 10 nMT3 is 123.2, 10 μM TMD is 24.8, 10 μM CPM was 256.1 and 100 μM MPA was 247.4.

(13) The number of branch points of GFAP positive cells is 1079.5 for the control group, 2616.5 for 10 nM Dex, 886 for 10 μM PMT, 774.1 for 10 nM E2, and 868.2 for 10 nM TCDD. 100.1M DHT is 2600.1, 100μM DEHP is 1149.7, 100pM BPA is 1514.1, 10nM PCB is 1607, 10nMT3 is 816.4, 10μM TMD is 160.3, 10μM CPM 1365.2 and 100μM MPA were 1211.2.

  Chemicals that promote neurite length (average total extension in one field) of MAP2-positive neurons compared to the control group (237214.07 μm) are 10 nM Dex (380272 μm), 10 μM PMT (362934 μm), 10 nM E2 (250986.2), 100 μM DEHP (224279 μm), 10 nM T3 (809916.1 μm), 10 μM TMD (251101.7 μm), 10 μM CPM (26316.2 μm), 100 μM MPA (4296.99.9 μm).

  On the other hand, the average value of the total elongation in one visual field of the GFAP glial-positive cell projection is promoted as compared with the Control group (43151.3 μm), 10 μMPMT (44386.1 μm), 10 nM DHT (1299023.1 μm), 100 μM DEHP (57664.5 μm), 100 pM BPA (61513.8 μm), 10 nM PCB (65410.1 μm), 100 nM CPM (597194.8 μm), 10 μM CPM (57194.8 μm), 100 μM MPA (4929.24 μm). .

  The total number of intersections of MAP2 positive neurons in one visual field was 10 nM Dex (1404.3) and 10 μM PMT (1404.3), which was larger than that in the Control group (1284.6). The average value of the total number of branch points of MAP2-positive neurons in one visual field was Control group (4615.6), 10 nM Dex (6548.9), 10 μM PMT (6230.7).

  Similarly, the average value of the total number of GFAP-positive glial cell cytoplasmic process intersections in one visual field is 10 nM Dex (407.9), 10 nM DHT (404.7) for the Control group (227.4). 100 pM BPA (302.7), 10 nM PCB (322.6), 10 μM CPM (256.1), 100 μM MPA (247.4), and the average value of the total number of branches in one field of view is 10 nM Dex (2616.5), 10 nM DHT (2600.1), 100 μM DEHP (1149.7), 100 pM BPA (1514.1), 10 nM PCB (1607) versus control group (1079.5) And 10 μM CPM (1365.2) and 100 μM MPA (1211.2).

Furthermore, when the average value of the total area in one visual field of the MAP2 positive neuron was examined, it was 10 nM E2 (732158.4 μm ² ) compared with the control group (601940.8 μm ² ). The average value of the total area in one field of view of the GFAP-positive glial cells, as compared to the Control group ^{(719544.9μm 2), 10μM PMT (} 955110μm 2), was 10nM E2 (831347.6μm ^2).

Next, a chemical substance that affects the morphology of the neurosphere is (1) a chemical substance whose average value of the total area in one field of neurosphere is larger than that of the control group (601939.769 μm ² ) is 10 nM E2 ( 7321588.4356 μm ² ). (2) The chemicals with a larger average value of the total number of neurospheres in one field of view than the control group (84.666667667) are 10 μM PMT (89.55555556), 100 nM TMD (86.0555556), ( 3) The average value of the roundness of the neurosphere was all close to a perfect circle (0.50 to 0.57) compared to the control group (0.4865) when exposed to chemical substances. ).

  (4) The chemical substance that promotes the average value of the total number of cells migrating from the neurosphere (total number of nuclei) in one visual field compared with the control group (14468.3889) is 10 nM Dex (35288.11111), 10 μM PMT (33720 pieces), 10 nM E2 (28991.11111 pieces), 10 nM DHT (32571.88889 pieces), 100 μM DEHP (333674.4444 pieces), 10 nM T3 (32269.8889 pieces), 10 μM TMD (22190.0556 pieces) They were 10 μM CPM (20452.1111) and 100 μM MAP (261777.5556).

  (5) Only 10 nM PCB (1565.881417 μm) was a chemical substance having a larger average circle circumference in one field of view compared to the control group (1551.69333 μm).

(6) The chemical substance in which the average value of the total nuclear area of the cell group in one visual field migrating from the neurosphere is larger than that of the control group (21294473.99 μm ² )
10 nM Dex (417172.276 μm ² ), 10 μM PMT (5146900.196 μm ² ), 10 nM E2 (3973015.467 μm ² ), 10 nM DHT (44848523.64 μm ² ), 100 μM DEHP (4591409.92 μm ² ), 10 nM 903 56 μm ² ), 10 μM TMD (3360761.74 μm ² ), 10 μM CPM (3104536.89 μm ² ), 10 μM MPA (38772487.964 μm ² ).

  In this way, graphing is possible based on the numerical information obtained by the multichannel image analyzer, so comparisons can be made for each chemical exposure group, but the correlation between morphological information and chemical substances is simple. It cannot be shown clearly. Therefore, TAO-Gen is a method for centrally arranging the morphological information and the variation information of the gene set (population) related to neuronal differentiation, and probabilistically estimating the dependency between the indices. The network between indicators for each chemical substance was expressed in a matrix using an algorithm. This matrix was obtained with different characteristics (classified) depending on the chemical substance.

  TAO-Gen (Theoretical Algorithm for Optimal Gene Interaction Networks) is a mathematical algorithm for creating an optimal gene interaction network. This technique is based on Bayes' theorem and is known. However, the present invention is the first application to the interrelation using multidimensional information by fusing morphological information and gene variation information.

6). Evaluation of experimental results (contrast method, judgment criteria, classification method, etc.)
The characteristic of each chemical substance obtained as a multi-information network diagram is that each index in the leftmost column is related to each index in the top row, as shown in FIGS. Black cells are shown as positive control relationships, and halftone cells are shown as negative control relationships. For example, focusing on the first line of the DMSO group, it shows that the AR gene has a positive control relationship with Esr2, and AR has a negative control relationship with Netrin1. In this way, it is possible to cover late effects from the initial exposure of chemical substances to a network diagram expressed by a 30 × 30 matrix based on 30 indices integrating 20 genes and 10 morphological parameters. It was. This network diagram is further divided into sub-categories (3 categories set and 3 categories related to genes and 4 sub-classes in total). Could be divided into chemicals.

With respect to the following blocks, the similarity frequency was calculated for each block with each cell being 1, and 90% or less was identified as different blocks based on 0.1% DMSO (see Table 3).
(AR, Esr1, Esr2, RARa, RARb, RARg) × Block 1 consisting of a matrix of (AR, Esr1, Esr2, RARa, RARb, RARg),
Block 2 comprising a matrix of (Cxcl12, Cxcr4, Itgb1, NGL1, NetrinG, Sema7A, Netrin1) × (AR, Esr1, Esr2, RARa, RARb, RARg),
(EphB, EphrinB, PlexinA, Robo2, Sema3A, Slit1, Unc5) × Block 3 consisting of a matrix of (AR, Esr1, Esr2, RARa, RARb, RARg),
(From EB_Area, EB_FormFactor, EB_Perimeter, EB_count, Nuc_Area, Nuc_count, Posi_Area, Branch_Point, Crossing_Point, Neut_Length, RA4, R1, RA, sr, RA1, sr, RA1, sr, RA1, sr
Block 5 comprising a matrix of (AR, Esr1, Esr2, RARa, RARb, RARg) × (Cxcl12, Cxcr4, Itgb1, NGL1, NetrinG, Sema7A, Netrin1),
(Cxcl12, Cxcr4, Itgb1, NGL1, NetrinG, Sema7A, Netrin1) × (Cxcl12, Cxcr4, Itgb1, NGL1, NetrinG, Sema7A, Netrin1) block 6,
(EphB, EphrinB, PlexinA, Robo2, Sema3A, Slit1, Unc5) × (Cxcl12, Cxcr4, Itgb1, NGL1, NetrinG, Sema7A, Netrin1) block 7,
(From EB_Area, EB_FormFactor, EB_Perimeter, EB_count, Nuc_Area, Nuc_count, Posi_Area, Branch_Point, Crossing_Point, Neut_Length, CNc4, CNc4, Nc_L, Nr_Count
A block 9 comprising a matrix of (AR, Esr1, Esr2, RARa, RARb, RARg) × (EphB, EphrinB, PlexinA, Robo2, Sema3A, Slit1, Unc5),
(Cxcl12, Cxcr4, Itgb1, NGL1, NetrinG, Sema7A, Netrin1) × (EphB, EphrinB, PlexinA, Robo2, Sema3A, Slit1, Unc5) block 10,
(EphB, EphrinB, PlexinA, Robo2, Sema3A, Slit1, Unc5) × (EphB, EphrinB, PlexinA, Robo2, Sema3A, Slit1, Unc5) block 11,
(From EB_Area, EB_FormFactor, EB_Perimeter, EB_count, Nuc_Area, Nuc_count, Posi_Area, Branch_Point, Crossing_Point, ph3, p2
(AR, Esr1, Esr2, RARa, RARb, RARg) × (EB_Area, EB_FormFactor, EB_Perimeter, EB_count, Nuc_Area, Nuc_count, Posi_Area, Branch_Point, Crow_Point, Crow_PointP
(Cxcl12, Cxcr4, Itgb1, NGL1, NetrinG, Sema7A, Netrin1) × (EB_Area, EB_FormFactor, EB_Perimeter, EB_count, Nuc_Area, Nuc_count, Nuc_count, P
(EphB, EphrinB, PlexinA, Robo2, Sema3A, Slit1, Unc5) x (from EB_Area, EB_FormFactor, EB_Perimeter, EB_count, Nuc_Area, Nuc_count, Poc_Count, Pos_P
Comprising (EB_Area, EB_FormFactor, EB_Perimeter, EB_count, Nuc_Area, Nuc_count, Posi_Area, Branch_Point, Crossing_Point, Neurite_Length) × (EB_Area, EB_FormFactor, EB_Perimeter, EB_count, Nuc_Area, Nuc_count, Posi_Area, Branch_Point, Crossing_Point, Neurite_Length) from the matrix of the block 16

  Table 3 Network block similarity based on DMSO group in network discrimination method

  12 chemical substances (2,3,7,8-tetrachlorinated-para-dioxin, 4 (OH) -2 ', 3,3', 4 ', 5'-penta-chlorobiphenyl, triiodothyronine Dexamethasone, 5α-dihydrotestosterone, 17β-estradiol, thalidomide, bis (2-ethylexyl), phthalic acid, permethrin, cyclopamine, bisphenol A, mesoprenic acid) and 12 different networks Could be classified. As a result, when testing chemical substances with unknown mechanism of action, similar exposure experiments were conducted on unknown chemical substances, and gene templates and cell morphology data were used to classify the network templates of existing chemical substances classified this time. It became possible to predict the impact by applying it. Specifically, the influence of the unknown substance can be predicted by creating a network matrix of the unknown substance and performing the same discrimination as the similarity of the network blocks shown in Table 3.

  By using the method of the present invention, the impact on embryonic programming can be evaluated. In addition, if a high-throughput analysis of late effects due to fetal exposure is performed using the method of the present invention, it is possible to use information for predicting biological (health) effects on a wide variety of chemical substances. This makes it possible to predict the impact on the living body (health) and to prevent damage from exposure to chemical substances.

Claims (13)

The following process:
A removable frame having 1020 uniform wells within a frame (10 × 10 mm) having a well diameter of 300 μm, a distance between wells (pitch) of 330 μm, and a well depth of 270 μm. 1 × 10 ⁵ cells / 250 μl of ES cell suspension per microsphere array was added using a 24 × 24 mm square frame-separated microsphere array, and the microspheres composed of ES cells were exposed to a nerve inducer, Forming a neurosphere precursor,
A method for creating a neuronal cell model, comprising differentiating cells constituting neurospheres into mature neurons by culturing the obtained neurosphere precursor in a nerve induction medium using a container for neuronal cell culture .   The method for creating a nerve cell system model according to claim 1, wherein the nerve cell culture container is an ornithine / laminin coated plate.   The method for creating a neuronal cell model according to claim 2, wherein all steps are completed in 20 days. The following process:
A removable frame having 1020 uniform wells within a frame (10 × 10 mm) having a well diameter of 300 μm, a distance between wells (pitch) of 330 μm, and a well depth of 270 μm. 1 × 10 ⁵ cells / 250 μl of ES cell suspension is added per microsphere array using a 24 × 24 mm square frame-separated microsphere array, and the microspheres composed of ES cells are brought into contact with the sample.
Separating a sample from the microsphere;
Exposing a part of the microsphere to a nerve inducer to form a neurosphere precursor, collecting total RNA from the remaining part, and measuring the expression level of the gene;
Creating a neuronal cell system model for differentiating the neurosphere-forming cells into mature neurons by culturing the obtained neurosphere precursor in a nerve induction medium using a neuron culture vessel,
A method for measuring the effect on embryonic programming comprising measuring the cell morphology of the resulting neural cell lineage model. The method for measuring the influence with respect to embryonic programming of Claim 4 whose said container for nerve cell cultures is an ornithine laminin coat plate. The above genes are:
One or more nuclear receptor genes selected from the group consisting of ERα, ERβ, AR, RARα and RARβ;
One or more susceptibility genes for schizophrenia selected from the group consisting of DTNBP1, NRG1, DAO, DAOA, RGS4, CAPON, PPP3CC, TRAR4, VCFS, COMT, PRODH, DHHC, ZDHHC8, DISC1 and GRM5,
One or more autism-related genes selected from the group consisting of Tsc1, Tsc2, Fmr1, Ube3a, Reln, Nlg3, Foxp2, Cntnap2, Slc6a4, Gabrb3, Mecp2 and En2,
One or more axon guidance related genes selected from the group consisting of EphrinB, EphB, Sema3A, PlexinA, Sema7A, Itgb1, Netrin1, Slit1, Robo2, Cxcl12, Cxcr4, NetrinG, NGL1 and Unc5,
One or more brain segmentation-related genes selected from the group consisting of Foxg1, Emx1, Emx2, Nkx2.1, Otx1, Otx2, En1, Gbx2, Hoxb1 and Hoxa2;
Snca, Uchl1, Apoe, Park7, Apbb1, Bcl2, Bcl2, Ube2l1, Ubqln1, Bax, Cdk5, Ubb, Als2, Gtf2a1, a group consisting of Base1, App, Psen1, Ide, Ccs, and Sod1Gp, and Sod1Gp, Sod1Gp Disease-related genes,
Nanog, Klf4, Zfp42, Pou5f1, Fgfr1, Nestin, Tuj1, Map2, Olig2, Gfap, Raf1, Atbf1, Pla2g6, Cdyl, Mapk3, Shc1, Hras1, Rps6k1, Hps1, Rps6k One or more neurodevelopmental genes selected from the group consisting of:
One or more Parkinson's disease-related genes selected from the group consisting of Th, Slc6a3, Snca, Ube1, Ubch7, Park2, Uch1, Park7, Casp9, Casp3 and Casp7, and / or App, Bace, Psen, ApoE, Ide, Mme 6. The method for measuring the effect on embryonic programming according to claim 5 , which is a combination with one or more Alzheimer's disease-related genes selected from the group consisting of Il1r1, Tnfrsf1a, Casp3 and Casp7. The cell morphology is neurosphere area, neurosphere roundness, neurosphere count, cell count, nucleus area, neurosphere circumference, neurite length, neurite intersection number, and neurite 6. The method for measuring the effect on embryonic programming according to claim 5 , which is the number of branch points. The method for measuring influence on embryonic programming according to claim 7 , wherein the measurement of the cell morphology is performed by a multi-channel cell image analyzer. The following process:
A removable frame having 1020 uniform wells within a frame (10 × 10 mm) having a well diameter of 300 μm, a distance between wells (pitch) of 330 μm, and a well depth of 270 μm. 1 × 10 ⁵ cells / 250 μl of ES cell suspension is added per microsphere array using a 24 × 24 mm square frame-separated microsphere array, and the microspheres composed of ES cells are brought into contact with the sample.
Separating a sample from the microsphere;
Exposing a part of the microsphere to a nerve inducer to form a neurosphere precursor, collecting total RNA from the remaining part, and measuring the expression level of the gene;
Creating a neuronal cell system model for differentiating the neurosphere-forming cells into mature neurons by culturing the obtained neurosphere precursor in a nerve induction medium using a neuron culture vessel,
Measurement of the cell morphology of the obtained neural cell system model Measurement of the obtained gene expression level and the measurement value of the cell morphology for a substance that is not treated with a sample or has a known effect on embryonic programming Contrast with value,
A method for assessing the impact on embryonic programming including: 10. The method for assessing impact on embryonic programming according to claim 9 , wherein the impact on embryonic programming is assessed using a Vegian network system. The following process:
A removable frame having 1020 uniform wells within a frame (10 × 10 mm) having a well diameter of 300 μm, a distance between wells (pitch) of 330 μm, and a well depth of 270 μm. 1 × 10 ⁵ cells / 250 μl of ES cell suspension is added per microsphere array using a 24 × 24 mm square frame-separated microsphere array, and the microspheres composed of ES cells are brought into contact with the sample.
Separating a sample from the microsphere;
Exposing a part of the microsphere to a nerve inducer to form a neurosphere precursor, collecting total RNA from the remaining part, and measuring the expression level of the gene;
Creating a neuronal cell system model for differentiating the neurosphere-forming cells into mature neurons by culturing the obtained neurosphere precursor in a nerve induction medium using a neuron culture vessel,
Measuring the cell morphology of the resulting neuronal cell line model, comparing the expression level of the gene and the measured value of the cell morphology with the measured value of a substance known to have an effect on embryonic programming, to determine the risk of lesions. A method of providing data for prediction. The following process:
A removable frame having 1020 uniform wells within a frame (10 × 10 mm) having a well diameter of 300 μm, a distance between wells (pitch) of 330 μm, and a well depth of 270 μm. 1 × 10 ⁵ cells / 250 μl of ES cell suspension is added per microsphere array using a 24 × 24 mm square frame-separated microsphere array, and the microsphere consisting of ES cells is brought into contact with the sample.
Separating a sample from the microsphere;
Exposing a part of the microsphere to a nerve inducer to form a neurosphere precursor, collecting total RNA from the remaining part, and measuring the expression level of the gene;
Creating a neuronal cell system model for differentiating the neurosphere-forming cells into mature neurons by culturing the obtained neurosphere precursor in a nerve induction medium using a neuron culture vessel,
A substance having an influence on embryonic programming, wherein the gene expression level and the measurement value of the cell morphology including the measurement of the cell morphology of the obtained neural cell model are compared with the measurement value when not treated with a sample. Method for screening. The method for screening according to claim 12 , wherein the substance having an influence on embryonic programming is an environmental chemical substance, a pharmaceutical product, or an agricultural chemical.

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