Embryo teratogenic substance screening and predicting model based on human induced pluripotent stem cells and construction method of embryo teratogenic substance screening and predicting model

Abstract

The invention discloses an embryo teratogenic substance screening prediction model based on human induced pluripotent stem cells and a construction method thereof, the embryo teratogenic substance screening prediction model is cells and / or cell aggregates, the cells are myocardial cells differentiated from the human induced pluripotent stem cells, and the cell aggregates are embryoid bodies. The embryo teratogenic substance screening prediction model uses human induced pluripotent stem cells to more accurately simulate the environment of human early embryonic development, avoids species difference of animal models, provides a relatively rapid and accurate screening platform, is suitable for high-throughput drug screening, and has good application prospects. Compared with the prior art, the kit has higher sensitivity and specificity, is helpful for eliminating compounds with potential reproductive toxicity in the early stage of drug development, and reduces the risk of later clinical test failure.

Description

Technical Field

[0001] The present invention belongs to the field of embryo teratogenicity evaluation of chemicals, and specifically relates to an embryo teratogen screening prediction model based on human induced pluripotent stem cells and a construction method thereof. Background Art

[0002] It is reported that about 8 million babies are born with congenital defects every year in the world, of which only 30% have clear causes. In the United States, 10% to 15% of congenital malformations are caused by inappropriate drugs, infections or other factors that interfere with normal embryonic development. Due to the difficulties in identifying and evaluating teratogens, up to 80% of the approved prescription drugs have unknown effects on pregnancy. Therefore, embryonic developmental toxicity evaluation is used to predict and evaluate the potential harm of drugs to embryonic or fetal development, which is of great significance in reducing and avoiding the occurrence of embryonic malformations or deaths and promoting the safety of medication during pregnancy. The commonly used method is in vivo animal experiments, but it requires a large amount of animals and scientific research funds. The alternative method not only conforms to the "3R" principle, but also controls confounding factors, which is convenient for exploring the mechanism of embryonic toxicity.

[0003] Induced pluripotent stem cells (iPSCs) are obtained by reprogramming somatic cells. Human iPSCs can avoid ethical and species differences, and show great potential in screening for reproductive developmental toxicity. The application of hiPSCs in embryonic developmental toxicity research is gradually gaining attention, but domestic research in this field is still in its infancy. Summary of the invention

[0004] The present invention provides a human induced pluripotent stem cell-based embryonic teratogen screening prediction model and a method for constructing the same. By combining the scheme of cardiomyocyte differentiation and embryoid body formation, a comprehensive evaluation of the potential effects of drugs on embryonic heart development and the entire embryonic development is achieved, and a new method for evaluating the embryonic teratogenicity of drugs is provided.

[0005] The present invention aims to construct a new model based on transcriptomics and molecular biology analysis methods to evaluate the toxicity of drugs on human induced pluripotent stem cells (hiPSCs) differentiated into cardiomyocytes and embryoid bodies (EBs) simulating the 14-day human embryonic development stage, and then predict the embryonic developmental toxicity of drugs.

[0006] The purpose of the present invention is to propose a method for constructing a teratogen evaluation model based on human induced pluripotent stem cell source, so as to provide a reference for subsequent potential teratogen screening.

[0007] In order to achieve the above object, the specific technical scheme of the present invention is as follows:

[0008] The present invention provides a cell and / or cell aggregate for screening embryo teratogens. The cell is a cardiomyocyte differentiated from human induced pluripotent stem cells, and the cell aggregate is an embryoid body.

[0009] In some specific embodiments, the cardiomyocyte biomarker is selected from troponin T (cTnT), actin, OCT4, MESP1, MYH6, NKX2.5, ACTN2 and TNNT2.

[0010] In some specific embodiments, the endoderm of the embryoid body expresses FOXA2 and GATA6, the mesoderm expresses Brachyury and MSX1, and the ectoderm expresses PAX6 and ZIC1.

[0011] The present invention also provides a method for constructing the cells and / or cell aggregates, the method comprising inducing differentiation culture of hiPSCs in a differentiation medium.

[0012] In some specific embodiments, the hiPSCs express SOX2 and NANOG.

[0013] In some embodiments, the differentiation culture medium is a cardiomyocyte differentiation medium or an embryoid body medium.

[0014] In some embodiments, the inducing differentiation culture further comprises culturing the hiPSCs in a cell culture medium such as mTeSR™ Plus.

[0015] In some specific embodiments, the cardiomyocyte differentiation medium is CardioEasy® complete human cardiomyocyte differentiation medium.

[0016] In some specific embodiments, the embryoid body culture medium is AggreWell™ EB Formation Medium.

[0017] The present invention also provides a cell model for screening or predicting embryo teratogens, and the cell model is constructed by the method.

[0018] The present invention also provides a method for screening embryo teratogens, the method comprising: co-incubating the cells and / or cell aggregates, or the cell model with a substance to be tested in a culture medium and obtaining detection data of an evaluation index; when the detection data deviates from a set threshold value of the evaluation index, judging that the substance to be tested has embryo toxicity.

[0019] In some embodiments, the test substances include abacavir, retinoic acid, caffeine, captopril, dabigatran, doxylamine, ethylene glycol, fluorouracil, glycolic acid, hydroxyurea, isotretinoin, methyl methacrylate (MEHP), methanol, methylmercury, metoclopramide, nilotinib, phenytoin, ramelteon, thalidomide, valproic acid, and methoxyacetic acid (MAA).

[0020] In some specific embodiments, the test substance is valproic acid.

[0021] In some embodiments, the evaluation index is selected from the group consisting of cell survival rate, cell proliferation rate, cardiomyocyte beating pattern, cell morphology, marker expression level and embryoid body area.

[0022] In some specific implementation schemes, the criteria for judgment are:

[0023] 1) For cardiomyocytes: after treatment with the test substance, the expression level of the biomarker gene is significantly different from that of the marker gene in the untreated group. When 0.01≤P<0.05, the test substance is judged to be mildly teratogenic; when 0.001≤P<0.01, the test substance is judged to be moderately teratogenic; when P<0.001, the test substance is judged to be severely teratogenic; otherwise, the test substance is judged to be non-teratogenic;

[0024] 2) For embryoid bodies: after treatment with the test substance, there is a significant difference in the expression levels of biomarker genes in the three germ layers of the embryoid bodies relative to the untreated group. When P<0.05, the test substance is judged to be mildly teratogenic; when P<0.01, the test substance is judged to be moderately teratogenic; when P<0.001, the test substance is judged to be severely teratogenic; otherwise, the test substance is judged to be non-teratogenic.

[0025] In some specific embodiments, the cardiomyocyte biomarker is selected from cTnT, Actinin, OCT4, MESP1, MYH6, NKX2.5, ACTN2 and TNNT2; the embryoid body marker gene is selected from FOXA2, GATA6, Brachyury, MSX1, PAX6 and ZIC1;

[0026] In some specific embodiments, the marker gene of the cardiomyocytes is TNNT2 or ACTN2; the marker gene of the embryoid body is GATA6, Bruchyury or PAX6.

[0027] In some specific embodiments, the embryoid body marker gene is Bruchyury.

[0028] The present invention also provides the use of the cells and / or cell aggregates in constructing a model for screening and predicting embryo teratogens.

[0029] The present invention also provides application of the cell model in screening or predicting embryo teratogens.

[0030] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0031] The reagents and raw materials used in the present invention are commercially available.

[0032] The positive and progressive effects of the present invention are:

[0033] In the present invention, human iPSC is used to construct an embryonic teratogen screening prediction model. The cell is a human skin fibroblast-derived cell with pluripotency. Human induced pluripotent stem cells are cultured in 3D to form embryoid bodies EBs and 2D culture to form cardiomyocytes as a drug screening model. EBs are very similar in structure and function to the egg cylinder period structure during early human embryonic development. It is an in vitro model system for studying mammalian embryonic development, and differentiated into contractile cardiomyocytes that simulate the early stages of human development. The effect of substances on early embryonic heart development can be studied. The experimental operation is simple, fast and convenient, which is conducive to large-scale raw material detection and screening work, and has high accuracy. The genes of hiPSC are homologous to humans, and cell morphology, biochemical markers and certain molecular events, such as gene expression, intracellular signal transduction, etc., are detected by microscopic observation, qPCR, immunofluorescence and other methods, which are used to compare the developmental toxicity of compounds to embryos, and are more reliable.

[0034] The present invention provides a novel model for predicting and evaluating the potential teratogenicity of drugs and other drugs on human embryonic development, which has the following advantages:

[0035] 1) The use of human induced pluripotent stem cells can more accurately simulate the environment of early human embryonic development and avoid species differences in animal models;

[0036] 2) Due to the use of in vitro culture system, the use of experimental animals is reduced, which complies with ethical requirements and reduces experimental costs and time;

[0037] 3) This system offers significant advantages over earlier EB systems, which relied heavily on the use of randomly sized mouse ESCs and EB aggregates. This system uses human induced pluripotent stem cells and strictly controlled and standardized EB culture conditions, providing a relatively rapid and accurate screening platform suitable for high-throughput drug screening.

[0038] 4) It can quantitatively analyze the changes in cell morphology and function caused by drugs, providing valuable data for early safety assessment of drugs;

[0039] 5) The repeatability and comparability of experimental results are ensured through standardized testing procedures;

[0040] Compared with the existing technology, the screening prediction model proposed in this patent has higher sensitivity and specificity, which helps to exclude compounds with potential reproductive toxicity in the early stages of drug development and reduce the risk of failure in later clinical trials. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the normal state of the hiPSC cells of the present invention.

[0042] Figure 2 Immunofluorescence staining was used to detect hiPSC pluripotency markers.

[0043] Figure 3 This is the embryoid body formation situation of the present invention.

[0044] Figure 4 This is the cardiomyocyte formation situation of the present invention.

[0045] Figure 5 Immunofluorescence staining was used to detect the cardiomyocyte markers troponin T (cTnT) and actin (Actinin).

[0046] Figure 6 The expression changes of cardiomyocyte-related genes (OCT4, MESP1, MYH6, NKX2.5, ACTN2, and TNNT2) within 10 days are shown in Figure 2. The data are expressed as mean ± standard deviation.

[0047] Figure 7The gene expressions of endoderm (FOXA2, GATA6), mesoderm (Brachyury, MSX1), and ectoderm (PAX6, ZIC1) in EBs cultured for 14 days were shown in Table 1. The data are presented as mean ± SD.

[0048] Figure 8 CCK8 cytotoxicity results of hiPSCs treated with graded concentrations of valproic acid.

[0049] Fig. 9 The expression of TNNT2 / ACTN2, a marker gene of cardiomyocytes, in samples 10 days after valproic acid administration. The data are expressed as mean ± standard deviation. There is statistical significance compared with the control condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

[0050] Fig.10 Changes in EB area of ​​samples after 14 days of valproic acid administration

[0051] Fig.11 The expression of EB marker genes GATA6 / Bruchyury / PAX6 in samples 14 days after valproic acid administration. Data are expressed as mean ± standard deviation. There is statistical significance compared with the control condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) DETAILED DESCRIPTION

[0052] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0053] Example

[0054] The embryo teratogen screening prediction model of the present invention comprises the following steps:

[0055] 1. Verification of hiPSC pluripotency

[0056] 1) Source of hiPSC cells: The cells in this study were derived from the cell line of Beijing Saibei Biotechnology Co., Ltd. (CA4027106), which were obtained by reprogramming normal human skin fibroblast-derived cells. Figure 1 shown.

[0057] 2) After cell digestion according to the iPSC subculture scheme, take an appropriate amount of cells and inoculate them in a 24-well plate pre-coated with Matrigel. After culturing for 2 to 3 days, discard the mTesr-Plus medium (Stemcell, 100-0276), wash twice with PBS, fix with 4% paraformaldehyde for 15 minutes at room temperature, and then wash three times with PBS; block the membrane with PBS containing 0.2% TritonX-100 and 3% BSA at room temperature for 60 minutes, and then incubate with fluorescent conjugated antibodies SOX2 (brand: abcam, ab305364) and NANOG (brand: abcam, ab196155) at 4°C overnight; the next day, wash three times with PBST (PBS+0.5% TWEEN) and stain the cell nucleus with Hoechst 33342 solution (10μg / ml) at room temperature in the dark for 5 minutes, add 1ml of PBS to each well of cells, and take pictures with a fluorescence microscope. The results are as follows Figure 2 shown.

[0058] 2. Differentiation of cardiomyocytes:

[0059] 1) Culture human iPSCs in mTeSR™ Plus. When the cells are in good condition and the cell confluence reaches 80%, prepare for differentiation;

[0060] 2) Aspirate the culture medium and wash once with PBS solution without calcium or magnesium;

[0061] 3) Add appropriate amount of CardioEasy® Human Cardiomyocyte Differentiation Medium I to each well of cells and culture in a constant temperature incubator for 48 hours.

[0062] 4) Aspirate human cardiomyocyte differentiation complete medium I, add PBS solution to each well to wash once, add CardioEasy® human cardiomyocyte differentiation complete medium II to each well, and culture in a constant temperature incubator for 48 hours;

[0063] 5) Aspirate human cardiomyocyte differentiation complete medium II, add PBS solution to each well to wash once, add CardioEasy® human cardiomyocyte differentiation complete medium III to each well, and culture in a constant temperature incubator;

[0064] 6) When the liquid turns yellow, replace it with fresh human cardiomyocyte differentiation complete medium III. After about 10 days, a large area of ​​beating cardiomyocytes will be observed under a microscope.

[0065] Cardiomyocyte Validation: Immunofluorescence Staining

[0066] 7) Wash the wells that have been filled with cells with PBS three times, 3 minutes each time;

[0067] 8) Fix the slides with 4% paraformaldehyde for 15 minutes, and wash the slides with PBS three times, 3 minutes each time;

[0068] 9) 0.5% Triton X-100 (prepared in PBS) permeabilization at room temperature for 20 min;

[0069] 10) Wash the slides with PBS three times for 3 min each time, dry the PBS with absorbent paper, add blocking solution to the slides, and block at room temperature for 40 min. During blocking, prepare the primary antibody (cTnT Anti-Cardiac Troponin T Antibody [1C11] (ab8295) / α-actin Anti-Sarcomeric Alpha Actinin Antibody (ab137346)) by diluting in antibody dilution buffer as indicated in the data sheet.

[0070] 11) Remove the blocking solution with absorbent paper, do not wash, add a sufficient amount of diluted primary antibody (1:50) to each slide and place in a humidified box, incubate at 4°C overnight;

[0071] 12) Rinse three times in 1X PBS, 5 minutes each time.

[0072] 13) Incubate samples in fluorochrome-conjugated secondary antibodies (Goat anti-mouse IgG H&L (AlexaFluor® 594) pre-adsorbed secondary antibody (ab150120) / Goat anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077)) diluted in antibody dilution buffer for 1 hour at room temperature in the dark.

[0073] 14) Rinse three times in 1X PBS, 5 minutes each time.

[0074] 15) The next day, wash three times with PBS, 3 minutes each time, add appropriate amount of 10μg / ml Hoechest to stain for 10 minutes, wash three times with PBS, and observe under a microscope. The results are as follows Figure 5 shown.

[0075] 16) qPCR detection markers

[0076] 17) First, samples were collected on days 3, 5, 7, and 10 during differentiation, total RNA was extracted, and then reverse transcribed into cDNA. qPCR was performed using the 2x Color SYBR Green gPCR Master Mix (ROX2 plus) kit, and relative gene expression was quantified using GAPDH as an internal reference. The results are shown in Figure 6As shown in the figure, during the differentiation of cardiomyocytes, the expression of different related genes showed specific temporal dynamic changes. In the initial stage, OCT4, as a marker of pluripotent stem cells, was initially expressed at a high level, but its expression decreased rapidly as differentiation progressed. MESP1 is a marker of early cardiomyocyte progenitor cells, and its expression increased significantly in the early differentiation stage, then reached a peak and gradually decreased. With the further differentiation and maturation of cardiomyocytes, cardiomyocyte-specific genes such as MYH6, NKX2.5, ACTN2, and TNNT2 began to be gradually expressed, among which NKX2.5, as a key cardiac transcription factor, reached a peak and continued to be expressed in the middle stage of differentiation. MYH6, ACTN2, and TNNT2 were significantly expressed in the late stage of differentiation, marking the gradual maturation of the structure and function of cardiomyocytes. The expression changes of these genes reflect the different stages and processes of cardiomyocyte differentiation, gradually transforming from pluripotent stem cells to mature cardiomyocytes.

[0077] 3. Embryoid Body Culture

[0078] iPS cell three-dimensional suspension culture as EB:

[0079] 1) Culture human iPSCs in mTeSR™ Plus. When the cells are in good condition and the cell confluence reaches 80%, directly digest the cells with GCDR digestion solution (Stemcell, 100-0485). Incubate at 37°C for 5-8 minutes before terminating the digestion.

[0080] 2) The digested cells were transferred to AggreWell™ EB Formation Medium supplemented with 10 μM Y-27632 for culture. We designed a strategy to mass produce thousands of human induced pluripotent stem cell spheres of uniform size, which can be synchronously differentiated in the presence and absence of suspected teratogens to generate EBs. Figure 3 shown.

[0081] 3) Collect cells by centrifugation and adjust to an appropriate cell density (about 5×10 4 ~2×10 5 cells / mL), and then inoculated into AggreWell™400 24-well plates for suspension culture;

[0082] 4) After culturing in embryoid body medium (AggreWell™ EB Formation Medium, stemcell, 05893) with Y-27632 induction factor for 24 hours, replace with new EB formation medium (AggreWell™ EB Formation Medium) without Y-27632 and continue culturing;

[0083] 5) In the next few days, observe the formation and development of embryoid bodies every day, and change half of the medium every day to provide fresh nutrients;

[0084] 6) After about 3 days, when the embryoid bodies reach the desired size and morphology, they are collected and transferred to a suitable culture dish for further culture;

[0085] 7) Collect embryoid bodies on days 1, 4, 8, and 14 for qPCR testing. Figure 7 As shown in the figure, during the 14-day culture of embryonic balls (EBs), the expression of different germ layer marker genes showed specific temporal dynamic changes. Endoderm marker genes FOXA2 and GATA6 were gradually upregulated in the early culture stage, indicating that the differentiation of endoderm cells was ongoing. FOXA2 was usually rapidly upregulated in the early stage of differentiation and then gradually decreased, while the expression of GATA6 was also significantly enhanced in the early stage and maintained at a high level during the subsequent culture process. The expression of mesoderm marker genes Brachyury and MSX1 increased significantly in the mid-culture stage. Brachyury, as a key gene for early mesoderm differentiation, was gradually upregulated to a peak during the differentiation induction process, and MSX1 gradually increased during the mid-differentiation process, reflecting the formation and development of mesoderm cells. The expression of ectoderm marker genes PAX6 and ZIC1 gradually increased in the late culture stage. PAX6 was significantly expressed in the late stage of ectoderm differentiation, indicating the formation of neural system progenitor cells and the beginning of neuronal differentiation, while the expression of ZIC1 also increased in the middle and late stages of ectoderm differentiation, marking the maturation of ectoderm cells. Overall, the expression changes of these germ layer marker genes depict the differentiation time course and dynamic development of endoderm, mesoderm, and ectoderm cells, reflecting the gradual establishment of the characteristics of different germ layer cells.

[0086] 4. Administration and effect evaluation of the drugs to be screened:

[0087] After obtaining stably beating cardiomyocytes and embryoid bodies with appropriate morphology, the drug to be screened (valproic acid) was administered to the two cell models respectively;

[0088] 4.1 Administration and effect evaluation of the drug to be screened, valproic acid, during differentiation of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes. The following are the detailed steps:

[0089] 1) Preparation before drug administration: First, prepare the stock solution of valproic acid, use sterile water as the solvent, and filter it through a filter membrane to ensure sterility. Before administering valproic acid, hiPSCs need to be in good growth state and have reached conditions suitable for differentiation, and the cell confluence should be close to 80%.

[0090] 2) Setting of valproic acid concentration: Taking into account the clinical application dose of valproic acid, as well as its effective concentration and cytotoxicity threshold in in vitro experiments, the concentration range of the drug was preliminarily set, and the final drug concentrations of 196.35 μM, 98.175 μM, and 49.875 μM were confirmed through CCK8 cytotoxicity experiments.

[0091] 3) Continuous administration throughout the differentiation process: A given concentration of valproic acid is added to the culture medium from the day of hiPSC differentiation and continues to be present throughout the differentiation process. Considering that the cell culture medium needs to be replaced every 2 days, the same concentration of valproic acid needs to be added each time the culture medium is replaced to keep the drug concentration constant.

[0092] 4) Evaluation of differentiation efficiency and cell survival: During drug treatment, the differentiation efficiency of cardiomyocytes was regularly evaluated by the expression levels of cardiomyocyte-specific markers, such as troponin TNNT2 and ACTN2 detected by qPCR.

[0093] 5) Observation of morphological changes: Observe and photograph the morphological changes of cells under a microscope, paying attention to whether the cells have irregular shapes, loss of contractile function, etc.

[0094] 6) Data recording and analysis: Real-time PCR was used to analyze the data using statistical software to compare the differences between the control group and the drug-treated group. The results were as follows: Fig. 9 As shown, 49.875 μM valproic acid was found to be severely teratogenic in the cardiomyocyte differentiation model.

[0095] 4.2 The administration and effect evaluation process of the drug to be screened, valproic acid, in embryoid bodies are described in detail as follows:

[0096] 1) Drug preparation: First, prepare a stock solution of valproic acid using sterile water as the solvent and filter through a membrane to ensure sterility.

[0097] 2) Concentration setting: Determine the concentration range of valproic acid based on previous drug dose screening experiments (such as cytotoxicity tests). Select several different concentration levels of non-cytotoxic concentrations, and confirm the final drug concentrations of 196.35μM, 98.175μM, and 49.875μM through CCK8 cytotoxicity experiments to evaluate the drug dose-dependent effect.

[0098] 3) Adjustment of culture conditions: hiPSCs should maintain a good growth state and sufficient confluence, usually about 80-90%, before differentiating into embryoid bodies (EBs).

[0099] 4) Drug administration: While hiPSCs are transferred to embryoid body culture conditions for three-dimensional suspension culture, a pre-set concentration of valproic acid is added. Cells are cultured in suspension in AggreWell™ to form embryoid bodies and are continuously exposed to the drug environment.

[0100] 5) Culture monitoring: During the entire embryoid body formation process, the morphological development and size changes of the cell clusters were observed and recorded daily. Half of the culture medium was replaced daily, and fresh valproic acid was added to maintain a stable drug concentration.

[0101] 6) Morphological and functional assessment: On the 14th day of embryoid body culture, the area of ​​the formed embryoid bodies was assessed.

[0102] 7) Differentiation index test: Evaluate the effect of valproic acid on the development of embryoid bodies and evaluate the drug effect by the area of ​​embryoid bodies.

[0103] 8) Data collection and analysis: GraphPad Prism statistical software was used to analyze the data and evaluate the differences between the treatment group and the control group. Fig.10 and Fig.11 It was found that the drug had moderate teratogenicity at a concentration of 49.875 μM. This concentration had no significant effect on the embryoid body area, but had a significant effect on gene expression, indicating that it had a significant teratogenic effect, which is consistent with the description in the drug instructions for valproic acid.

[0104] 5. The criteria for determining embryonic teratogenicity using the functional indicators are:

[0105] 1) For cardiomyocyte differentiation: when the expression of marker genes between the drug-treated group and the blank control group is P<0.05, the drug causes mild teratogenicity, otherwise, there is no damage; when the expression of marker genes between the drug-treated group and the blank control group is P<0.01, the drug causes moderate teratogenicity; when the expression of marker genes between the drug-treated group and the blank control group is P<0.001, the drug causes severe teratogenicity. The most intuitive manifestation of the effect of drugs on teratogenicity is the change in gene expression. Therefore, the change in gene expression can intuitively reflect the degree of inhibition of differentiation by drugs.

[0106] 2) For embryoid bodies: drugs that affect the expression of genes in a certain germ layer and promote the expression of other germ layers are also considered to be teratogenic. When the P value between the expression of a gene in the three germ layers of embryoid bodies between the drug-treated group and the blank control group is <0.05, the drug causes mild teratogenicity, otherwise, there is no damage; when the P value between the expression of a gene in the three germ layers of the drug-treated group and the blank control group is <0.01, the drug causes moderate teratogenicity; when the P value between the expression of a gene in the three germ layers of the drug-treated group and the blank control group is <0.001, the drug causes severe teratogenicity.

Claims

1. A cell and / or cell aggregate for embryo teratogen screening, characterized in that: The cells are cardiomyocytes differentiated from human induced pluripotent stem cells, and the cell aggregates are embryoid bodies.

2. The cell and / or cell aggregate according to claim 1, characterized in that The cardiomyocyte biomarker is selected from troponin T (cTnT), actin, OCT4, MESP1, MYH6, NKX2.5, ACTN2 and TNNT2; And / or, the endoderm of the embryoid body expresses FOXA2 and GATA6, the mesoderm expresses Brachyury and MSX1, and the ectoderm expresses PAX6 and ZIC1.

3. A method for constructing the cell and / or cell aggregate according to claim 1 or 2, characterized in that: The method comprises inducing differentiation culture of hiPSCs in a differentiation medium; Preferably, the hiPSC expresses SOX2 and NANOG.

4. The method according to claim 3, characterized in that The culture medium for differentiation culture is a cardiomyocyte differentiation culture medium or an embryoid body culture medium; and / or, before the induction differentiation culture, the hiPSCs are cultured in a cell culture medium such as mTeSR™ Plus.

5. The method according to claim 4, characterized in that The cardiomyocyte differentiation medium is CardioEasy® human cardiomyocyte differentiation complete medium; and / or, the embryoid body medium is AggreWell™ EB Formation Medium.

6. A cell model for screening or predicting embryonic teratogens, characterized in that: The cell model is constructed by the method according to any one of claims 3 to 5.

7. A method for screening embryo teratogens, characterized in that: The method comprises: co-incubating the cells and / or cell aggregates as described in claim 1 or 2, or the cell model as described in claim 6, with a substance to be tested in a culture medium and obtaining detection data of an evaluation index; when the detection data deviates from a set threshold value of the evaluation index, judging that the substance to be tested has embryotoxicity.

8. The method according to claim 7, characterized in that The evaluation index is selected from the group consisting of cell survival rate, cell proliferation rate, cardiomyocyte beating pattern, cell morphology, marker expression level and embryoid body area; Preferably, the judgment criteria are: 1) For cardiomyocytes: After the test substance is treated, the expression level of the biomarker gene is significantly different from the expression level of the marker gene in the untreated group. When 0.01≤P<0.05, the test substance is judged to be mildly teratogenic; when 0.001≤P<0.01, the test substance is judged to be moderately teratogenic; When P<0.001, the substance to be tested is judged to be severely teratogenic; otherwise, the substance to be tested is judged to be non-teratogenic; 2) For embryoid bodies: After the test substance is treated, the expression levels of biomarker genes in the three germ layers of the embryoid bodies are significantly different from those in the untreated group. When P<0.05, the test substance is judged to be mildly teratogenic; when P<0.01, the test substance is judged to be moderately teratogenic; When P<0.001, the substance to be tested is judged to be severely teratogenic; otherwise, the substance to be tested is judged to be non-teratogenic; More preferably, the biomarker of cardiomyocytes is selected from cTnT, Actinin, OCT4, MESP1, MYH6, NKX2.5, ACTN2 and TNNT2; the marker gene of embryoid body is selected from FOXA2, GATA6, Brachyury, MSX1, PAX6 and ZIC1; Further preferably, the marker gene of the cardiomyocytes is TNNT2 or ACTN2; the marker gene of the embryoid body is GATA6, Bruchyury or PAX6, preferably Bruchyury.

9. Use of the cells and / or cell aggregates as described in claim 1 or 2 in constructing a model for screening and predicting embryo teratogens.

10. Use of the cell model as claimed in claim 6 in screening or predicting embryo teratogens.

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