Toxicity evaluation system based on human pluripotent stem cell model with introduced fluorescent marker for real-time monitoring of cytoplasmic phase separation phenomenon

Abstract

The present invention relates to a toxicity evaluation system based on a human pluripotent stem cell model with an introduced fluorescent marker for the real-time monitoring of cytoplasmic phase separation phenomenon, in which a CRISPR-Cas9 system was used to establish human pluripotent stem cells with permanent endogenous expression of a fluorescent marker at the C-terminal of the G3BP1 gene, which is one of the representative proteins involved in phase separation phenomena, and it can be confirmed that the human pluripotent stem cells have no problem with differentiation into three germ layers, and undergo phase separation phenomena, particularly, formation of stress granules, in real time upon treatment with a test substance. Therefore, this system can be effectively used as a technology that enables real-time evaluation of toxicity resulting from exposure to various harmful factors, after differentiating human pluripotent stem cells into various cells and organoids that constitute the human body.

Description

A toxicity assessment system based on a human pluripotent stem cell model with a fluorescent marker for real-time monitoring of cytoplasmic phase separation.

[0001] The present invention relates to a toxicity assessment system based on a human pluripotent stem cell model into which a fluorescent marker capable of monitoring cytoplasmic phase separation phenomenon in real time is introduced.

[0002] This research was supported by the 'Development of a real-time lung toxicity assessment method for mixtures containing household chemical products using human lung organoids with fluorescent labels' project (Project No.: 1485019341, RS-2022-KE002021), a project of the Korea Environmental Industry & Technology Institute under (or affiliated with) the Ministry of Environment's household chemical product safety management technology development project.

[0003] Alternative animal testing (AAT) is a technology that replaces laboratory animals to evaluate the efficacy and toxicity of chemicals. It is considered a promising approach to overcome the inconsistencies between animal testing and clinical trials due to species differences. Furthermore, the technology holds significant future potential, with potential applications in regenerative therapeutics, personalized medicine, and disease modeling.

[0004] As interest in laboratory animal welfare grows, particularly in advanced economies, a growing movement is underway within related industries to discourage the use of laboratory animals. Beginning with the UK, at least 23 countries around the world have enacted laboratory animal protection laws. Europe has banned the sale of cosmetics tested on animals within the EU since 2013. In the US, state governments like New York and California are regulating the sale of cosmetics tested on animals.

[0005] Recently, various in vitro toxicity evaluation methods using differentiated cells derived from human stem cells have been known, such as measuring the survival rate by pharmaceuticals and chemicals and measuring ROS, but an evaluation method that can monitor toxicity reactions based on fluorescence images in real time without destroying cells has not been reported.

[0006] Therefore, to enable rapid and accurate toxicity assessment following exposure to pharmaceuticals and chemicals, it is necessary to develop a novel human normal cell model system for toxicity assessment that can monitor the expression of acute toxicity markers in real time.

[0007] Meanwhile, Korean Patent No. 1455244 discloses a quantitative cytotoxicity evaluation system and method using an AFM and a probe-type sensor, and Korean Patent No. 2466092 discloses a human liver chimeric non-human animal having a defective P450 oxidoreductase and a method for using the same. However, there is no disclosure at all regarding a toxicity evaluation system based on a human pluripotent stem cell model with a fluorescent marker that can monitor the cytoplasmic phase separation phenomenon of the present invention in real time.

[0008] The present invention was derived from the above-mentioned needs, and provides a human pluripotent stem cell system in which a fluorescent marker is introduced immediately before the stop codon at the 3' end of the G3BP1 gene using the CRISPR-Cas9 system for toxicity evaluation based on a human pluripotent stem cell model, and verifies the differentiation ability of the human pluripotent stem cell into the three germ layers, and completes the present invention by confirming the phase separation phenomenon after differentiating the human pluripotent stem cell into a specific cell or a specific organoid and then treating it with a test substance for toxicity evaluation (a test substance causing toxicity).

[0009] To solve the above problem, the present invention provides human pluripotent stem cells into which a fluorescent reporter for toxicity assessment is introduced, which detects phase separation phenomenon in real time.

[0010] In addition, the present invention provides a cell differentiated from the human pluripotent stem cell.

[0011] Additionally, the present invention provides an organoid differentiated from the human pluripotent stem cell.

[0012] In addition, the present invention provides a composition for toxicity evaluation comprising the human pluripotent stem cell, a cell differentiated therefrom, or an organoid.

[0013] In addition, the present invention provides a composition for screening for toxic substances comprising the human pluripotent stem cells, cells differentiated therefrom, or organoids.

[0014] In addition, the present invention provides a method for producing human pluripotent stem cells into which a fluorescent reporter for toxicity evaluation has been introduced, comprising the step of introducing a fluorescent marker to the 3'-end of the G3BP1 gene using a CRISPR-Cas9 system.

[0015] In addition, the present invention

[0016] 1) a step of treating the human pluripotent stem cells, cells differentiated therefrom, or organoids with a test substance for toxicity evaluation; and

[0017] 2) A method for screening toxic substances is provided, including a step of predicting toxic substances by confirming a real-time phase separation phenomenon after the above step 1).

[0018] The present invention relates to a toxicity assessment system based on a human pluripotent stem cell model that introduces a fluorescent marker for real-time monitoring of a cytoplasmic phase separation phenomenon. By using the CRISPR-Cas9 system, human pluripotent stem cells that permanently and endogenously express a fluorescent marker at the C-terminal portion of the G3BP1 gene, which is one of the representative proteins constituting the phase separation phenomenon, were established, and it was confirmed that the human pluripotent stem cells differentiated into three germ layers without any abnormalities, and when treated with a test substance, the phase separation phenomenon, specifically, the formation of stress granules, occurred in real time.

[0019] In addition, the human pluripotent stem cells can be used to produce various cells and organoids that constitute the human body, and can be usefully utilized as a technology for evaluating toxicity in real time due to exposure to various harmful factors.

[0020] In particular, in the case of human induced pluripotent stem cells, the vector transfection efficiency is significantly low, so it is a very difficult technology to accurately introduce a fluorescent gene into the target gene location. A system that can stably maintain and culture normal human induced pluripotent stem cells with a cytoplasmic phase separation marker fluorescent label that can differentiate into all cells of the human body, and monitor in real time the formation of stress granules, a phase separation phenomenon that appears very quickly when exposed to pharmaceuticals, chemicals, environmental hazards, and biological hazards, is of very high technological prowess.

[0021] Figure 1 is a schematic diagram of a CRISPR / Cas9-based gene insertion (knock-in) system for introducing a fluorescent marker into a phase separation marker gene (G3BP1).

[0022] Figure 2 shows the information on the gene insertion vector (knock-in vector, KI vector) constructed to introduce a fluorescent marker into the phase separation marker gene (G3BP1) and the results of extracting plasmid DNA and confirming its size. M is a size marker.

[0023] Figure 3 shows the results of analyzing the expression of pluripotency markers (OCT3 / 4, SOX2, NANOG) using immunofluorescence to confirm whether human induced pluripotent stem cells (iPSCs) into which a specific fluorescent marker for the phase separation marker gene (G3BP1) of the present invention has been introduced possess stem cell characteristics. Nuclear counterstaining was performed using Hoechst33342.

[0024] Figure 4 shows the results of verifying the differentiation potential of human induced pluripotent stem cells into the three germ layers, into which a specific fluorescent marker of the phase separation marker gene (G3BP1) of the present invention has been introduced. A is the result of morphological analysis under a microscope, B is the result of confirming the mRNA expression level of the three germ layer markers (PAX6, Nestin, Desmin, α-SMA, SOX17, GATA4) through qPCR, and C is the result of confirming the protein expression of the three germ layer markers (Nestin, α-SMA, SOX17) through immunofluorescence.

[0025] Figure 5 shows the results of confirming the phase separation phenomenon (stress granule formation) according to NaAsO2 treatment in human induced pluripotent stem cells into which the phase separation marker gene (G3BP1) specific fluorescent marker of the present invention has been introduced. The control group is a group of human induced pluripotent stem cells into which the phase separation marker gene (G3BP1) specific fluorescent marker of the present invention has been introduced and which have not been treated with NaAsO2.

[0026] Figure 6 shows the results of analyzing the expression of a cardiomyocyte-specific marker by inducing differentiation into cardiomyocytes from human induced pluripotent stem cells into which a specific fluorescent marker for the phase separation marker gene (G3BP1) of the present invention has been introduced. A shows the results of confirming the protein expression of the fluorescent marker introduced into the G3BP1 gene through fluorescence microscopy. B shows the results of confirming the protein expression of the cardiomyocyte-specific marker (cTnT) through flow cytometry.

[0027] Figure 7 shows the results of analyzing the expression of lung cell-specific markers by inducing differentiation into lung cells from human induced pluripotent stem cells into which a specific fluorescent marker for the phase separation marker gene (G3BP1) of the present invention has been introduced. A shows the results of confirming the protein expression of the fluorescent marker introduced into the G3BP1 gene through a fluorescence microscope and the protein expression of the lung cell-specific marker (NKX2.1) using immunofluorescence. Nuclear counterstaining was performed using Hoechst33342. B shows the results of confirming the protein expression levels of lung cell-specific markers (CPM and MUC1) using flow cytometry.

[0028] Figure 8 shows the results of analyzing the expression of lung-specific markers and phase separation phenomenon (stress granule formation) according to NaAsO2 treatment by inducing differentiation into lung organoids from human induced pluripotent stem cells into which a phase separation marker gene (G3BP1) specific fluorescent marker of the present invention has been introduced. A is the result of confirming the morphology of lung organoids through microscopic observation. B is the result of confirming the protein expression of the lung-specific marker (NKX2.1) by immunofluorescence. Nuclear counterstaining was performed using Hoechst33342. C is the result of confirming the phase separation phenomenon (stress granule formation) according to NaAsO2 treatment. Control is a group of lung organoids derived from human induced pluripotent stem cells into which a phase separation marker gene (G3BP1) specific fluorescent marker of the present invention has been introduced and not treated with NaAsO2.

[0029] In order to achieve the purpose of the present invention, the present invention provides human pluripotent stem cells into which a fluorescent reporter for toxicity evaluation is introduced, which detects a phase separation phenomenon in real time.

[0030] The above human pluripotent stem cells are preferably, but not limited to, embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0031] The term 'phase separation phenomenon' of the present invention refers to a process in which macromolecules within a cell are separated into a dense phase and a relatively dilute phase by stimulation, and the phase separation phenomenon of the present invention is preferably characterized by the formation of stress granules.

[0032] The fluorescent reporter for the above toxicity evaluation can be any protein that expresses fluorescence, and is preferably one selected from the group consisting of firefly luciferase, renilla luciferase, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), mCherry, and DsRed, but is not limited thereto.

[0033] In one embodiment of the present invention, the human pluripotent stem cell into which the fluorescent reporter for toxicity evaluation is introduced may be one in which a fluorescent marker is introduced into the G3BP1 gene using the CRISPR-Cas9 system, preferably one in which a fluorescent marker is introduced immediately before the stop codon at the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system, and more preferably one in which a fluorescent marker is introduced into the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system, such as firefly luciferase, renilla luciferase, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (ENFP), Any one fluorescent marker selected from the group consisting of, but not limited to, EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), mCherry, and DsRed is introduced.

[0034] The 'toxicity assessment' of the present invention is to investigate the risk to humans of test substances [pesticides, pharmaceuticals, household chemicals, industrial chemicals, other environmental pollutants, or biological hazards (bacteria and viruses)], and to evaluate the degree of risk to each of cells, spheroids, or organoids differentiated from human pluripotent stem cells into which a fluorescent reporter for toxicity assessment, which detects the phase separation phenomenon of the present invention in real time, has been introduced.

[0035] In addition, the present invention provides a cell differentiated from the human pluripotent stem cell.

[0036] The above differentiated cells can be differentiated into any human pluripotent stem cell of the present invention.

[0037] In one embodiment of the present invention, the human pluripotent stem cell can differentiate into any one normal or abnormal cell selected from lung cells, heart cells, blood cells, liver cells, pancreatic cells, brain cells, small intestinal cells, colon cells, stomach cells, kidney cells, and skin cells, but is not limited thereto.

[0038] Additionally, the present invention provides an organoid differentiated from the human pluripotent stem cell.

[0039] The above organoids can be differentiated into any type of organ if they are derived from human pluripotent stem cells.

[0040] In one embodiment of the present invention, the organoid may be an organoid that mimics any normal or abnormal tissue selected from among small intestine, large intestine, liver, pancreas, kidney, brain, lung, heart, hair root, and skin, but is not limited thereto.

[0041] In addition, the present invention provides a composition for toxicity evaluation comprising the human pluripotent stem cell, a cell differentiated therefrom, or an organoid.

[0042] The above toxicity assessment may be, but is not limited to, a toxicity assessment caused by any of the following: pesticides; household chemical products or industrial chemicals; and biological hazards including bacteria or viruses. A preferred example of the household chemical product or industrial chemical is, but is not limited to, sodium arsenite.

[0043] The composition for the above toxicity evaluation is characterized in that after producing cells or organoids differentiated from human pluripotent stem cells, the test substance is treated, and the degree of stress granule formation is measured in real time using a fluorescent reporter, and the degree of stress granule formation can be confirmed using a microscope.

[0044] In addition, the present invention provides a composition for screening for toxic substances comprising the human pluripotent stem cells, cells differentiated therefrom, or organoids.

[0045] The above composition can be used to screen for substances that cause toxicity by producing cells or organoids differentiated from human pluripotent stem cells, treating the cells with a test substance to determine whether they cause toxicity, and then checking the degree of stress granule formation in real time.

[0046] The above toxic substances are preferably, but not limited to, pesticides; household chemical products or industrial chemicals; and biological hazards including bacteria or viruses.

[0047] In addition, the present invention provides a method for producing human pluripotent stem cells into which a fluorescent reporter for toxicity evaluation has been introduced, comprising the step of introducing a fluorescent marker to the 3'-end of the G3BP1 gene using a CRISPR-Cas9 system.

[0048] The above manufacturing method may include a step of transfecting stem cells with a CRISPR-Cas9-sgRNA vector including sgRNA; and a gene insertion (Knock-in) vector for introducing a fluorescent marker into the G3BP1 gene; and introducing a fluorescent marker into the 3'-end of the G3BP1 gene; and specifically, the above manufacturing method preferably includes a step of introducing a fluorescent marker into the 3'-end of the G3BP1 gene using a CRISPR-Cas9 system including sgRNA of SEQ ID NO: 1, but is not limited thereto.

[0049] In one embodiment of the present invention, a CRISPR-Cas9-sgRNA vector for CRISPR-Cas9-based gene editing and a gene insertion (Knock-in) vector for introducing a fluorescent marker into the phase separation marker (G3BP1) gene were transfected into stem cells, thereby establishing human pluripotent stem cells that permanently endogenously express a fluorescent marker immediately before the 3'-terminal stop codon of the G3BP1 gene through CRISPR-Cas9-based gene insertion (Knock-in).

[0050] There is no particular limitation on the type of Cas9 vector as long as it is a vector that expresses the Cas9 protein, and the pX330 plasmid is preferred.

[0051] In order to produce a gene insertion (knock-in) vector for introducing a fluorescent marker into the phase separation marker (G3BP1) gene, it is preferable to design a 5' homology arm (5'HA) of 500 to 600 bp and a 3' homology arm (3'HA) of 700 to 900 bp based on the stop codon sequence located in exon 12 of the G3BP1 gene, and to insert the fluorescent gene therebetween, and more preferably, a 5' homology arm (5'HA) of 550 bp and a 3' homology arm (3'HA) of 800 bp based on the stop codon sequence located in exon 12 of the G3BP1 gene, and to insert the fluorescent gene therebetween, but the present invention is not limited thereto.

[0052] In one embodiment of the present invention, a pDSRed vector may be used to produce a gene insertion (knock-in) vector for introducing a fluorescent marker into a phase separation marker (G3BP1) gene, but is not limited thereto.

[0053] In addition, the present invention

[0054] 1) a step of treating the human pluripotent stem cells, cells differentiated therefrom, or organoids with a test substance for toxicity evaluation; and

[0055] 2) A method for screening toxic substances is provided, including a step of predicting toxic substances by confirming a real-time phase separation phenomenon after the above step 1).

[0056] The cells differentiated from the human pluripotent stem cells of the above step 1) are any one normal or abnormal cell selected from lung cells, heart cells, blood cells, liver cells, pancreatic cells, brain cells, small intestine cells, large intestine cells, stomach cells, kidney cells, and skin cells, but are not limited thereto.

[0057] The above abnormal cells may be any disease model. For example, the above abnormal cells may be lung cancer cells, liver cancer cells, stomach cancer cells, colon cancer cells, or fatty liver cells, but are not limited thereto.

[0058] The organoid of the above step 1) may be an organoid that mimics any normal or abnormal tissue selected from among small intestine, large intestine, liver, pancreas, kidney, brain, lung, heart, hair root, and skin, but is not limited thereto.

[0059] The organoids mimicking the above-mentioned abnormal tissues can be any disease model. Examples include, but are not limited to, organoids mimicking liver cancer, stomach cancer, colon cancer, fatty liver disease, fibrotic tissue, and genetic disease-specific organoid models.

[0060] The test substance for toxicity evaluation in step 1) above refers to any substance that is expected to induce phase separation, specifically stress granule formation, when treated with organoids or differentiated cells.

[0061] The step of confirming the phase separation phenomenon in the above step 2) is a step of confirming the phase separation phenomenon, specifically the formation of stress granules, by comparing it with a control group that was not treated with the test substance, and the method of confirming the phase separation phenomenon is not particularly limited as long as it is a method that can confirm the formation of stress granules with the naked eye.

[0062]

[0063] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0064]

[0065] Example 1. Production of a CRISPR / Cas9-based gene insertion vector (knock-in vector) for introducing a fluorescent marker into the phase separation marker (G3BP1) gene.

[0066] A schematic diagram of a CRISPR / Cas9-based gene insertion (Knock-in) system for introducing a fluorescent marker into the phase separation marker gene (G3BP1) of the present invention is as disclosed in Fig. 1.

[0067]

[0068] 1) Production of CRISPR / Cas9-sgRNA vector for CRISPR / Cas9-based gene editing

[0069] To construct a CRISPR / Cas9-sgRNA vector targeting G3BP1 exon 12, a DNA oligomer complementary to sgRNA was synthesized. The sequence of sgRNA is shown in SEQ ID NO: 1 in Table 1 below.

[0070] The pX330 plasmid was used as the Cas9 vector, and this was digested with the restriction enzyme BbsI to obtain the pX330 / BbsI vector. Afterwards, the obtained annealed sgRNA was ligated to the pX330 / BbsI vector, transformed, and cloned. Afterwards, the culture was plated on LB agar medium containing ampicillin and cultured at 37°C. Colony formation was confirmed, and DNA was extracted from each colony and sequenced to confirm that the sgRNA had entered the correct position in the vector.

[0071] Sequence number sgRNA sequence 15'-CGACGAGATAATCGCCTTCG-3'

[0072]

[0073] 2) Production of a gene insertion vector for introducing a fluorescent marker into the phase separation marker (G3BP1) gene

[0074] To produce a targeting vector to induce endogenous expression by introducing a fluorescent marker into the 3'-terminal of the G3BP1 gene, a 5' homology arm (5'HA) of 550 bp and a 3' homology arm (3'HA) of approximately 800 bp were designed based on the stop codon sequence located in exon 12 of the G3BP1 gene, and the fluorescent marker gene was inserted between them.

[0075] Specifically, a donor vector and insert were secured for the construction of a G3BP1-GFP gene knock-in vector. The primer sequences used to construct the homology arm vector are as disclosed in SEQ ID NOs: 2 to 7 of Table 2 below.

[0076] The promoter-less pDsRed-Express2-1 plasmid was used as a donor vector, and the DsRed-Express 2 region was removed by digestion with BglII and NotI, thereby obtaining the pDsRed / BglII / NotI vector.

[0077] To produce the insert, three fragments, G3BP1 5'HA with BglII restriction enzyme sequence, G3BP1 3'HA with GFP and NotI restriction enzyme sequences, were obtained through PCR, and then ligated through overlap extension PCR to obtain the final BglII-G3BP1 5'HA_GFP_G3BP1 3'HA-NotI insert.

[0078] Afterwards, the insert (BglII-G3BP1 5'HA_GFP_G3BP1 3'HA-NotI) was ligated into the constructed vector (pDsRed / BglII / NotI), transformed, and cloned. Afterwards, the culture was spread on LB agar medium containing kanamycin and cultured at 37°C. After confirming the formation of colonies, DNA was extracted from each colony to obtain a G3BP1-GFP gene insertion vector (G3BP1-GFP knock-in vector) as shown in Fig. 2. Then, sequence analysis was performed to confirm that the sequence of the obtained vector was 100% identical to the intended sequence.

[0079] SEQ ID NO: primer sequence 2BglII-G3BP1 5' HA forward5'-ATATAGATCTCAATGGCGTGATCTTGGCT-3'3G3BP1 5' HA reverse5'-CTGTCGTGGCGCAAGC-3'4G3BP1 5' HA-GFP forward5'-AAGGGGGCTTGCGCCACGACAGATGGTGAGCAAGGGCGA-3'5GFP-G3BP1 3' HA reverse5'-GCATGAAGATCCATGAAGATTTACTTGTACAGCTCGTCCATG-3'6G3BP1 3' HA forward5'-ATCTTCATGGATCTTCATGCAG-3'7G3BP1 3' HA-NotI reverse5'-TAGCGGCCGCAGGCACAACAGTTTTGCTC-3'

[0080]

[0081] Example 2. Analysis of pluripotency marker expression in human pluripotent stem cell lines into which a phase separation marker (G3BP1) gene-specific fluorescent marker has been introduced.

[0082] To confirm whether the human induced pluripotent stem cell line CMC-hiPSC-009 (hereinafter referred to as CMC9_G3BP1-EGFP), into which a G3BP1 target fluorescent marker was introduced, possesses unique stem cell properties, the expression of pluripotency markers (OCT3 / 4, SOX2, NANOG) was analyzed using immunofluorescence.

[0083] First, to introduce a phase separation marker gene-specific fluorescent label into the human induced pluripotent stem cell line CMC-hiPSC-009, 8 × 10 cells were transfected using Lipofectamine™ Stem Transfection Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol. 4 Cells were transfected with 2 μg of the knock-in construct (CRISPR / Cas9-sgRNA vector + G3BP1-CFP gene insertion vector) per cell. After transfection, cells were cultured in a humidified 37°C, 5% CO2 incubator until analysis.

[0084] Afterwards, human induced pluripotent stem cell line CMC-hiPSC-009 (CMC9_G3BP1-EGFP) with G3BP1 target fluorescent marker was introduced into Matrigel-coated 18-well μ-slides at 3 × 10 per well. 3 Cells were seeded and cultured in a humidified 37°C, 5% CO2 incubator for 3 days. The culture medium used was mTeSR-1, and fresh culture medium was replaced every 24 hours. After culturing for 3 days, the cells were washed three times with DPBS, fixed for 20 minutes with 4% (v / v) PFA fixative, washed three times with DPBS, and permeabilized with 0.1% TritonX-100 for 10 minutes at room temperature. After that, the cells were washed three times with DPBS, and blocked with 1% BSA for 1 hour at room temperature.

[0085] For OCT3 / 4 and SOX2 staining, antibodies (#SC021, NL557-conjugated SOX2 and NL637-conjugated Oct-3 / 4) were diluted 1:10 in DPBS solution containing 2% BSA and 0.1% Triton X-100, incubated at room temperature for 3 hours, washed three times with DPBS, and then nuclear counterstaining was performed by incubating with Hoechst 33342 diluted 1:1,000 in DPBS for 20 minutes at room temperature.

[0086] For NANOG staining, the primary antibody (#4903S, cell signaling) was diluted 1:200 in DPBS containing 2% BSA and 0.1% Triton X-100 and incubated overnight at 4°C. The membrane was washed three times with DPBS, and then the secondary antibody (#A21447, Goat anti-Mouse IgG (H+L), Alexa Fluor™ 647) was diluted 1:1,000 in DPBS containing 2% BSA and 0.1% Triton X-100 and incubated at room temperature for 3 hours. After washing three times with DPBS, Hoechst 33342 was diluted 1:1,000 in DPBS and incubated at room temperature for 20 minutes to perform nuclear counterstaining.

[0087] Stem cells after immunofluorescence staining were analyzed for pluripotency marker expression using a confocal microscope.

[0088] As a result, it was confirmed that the human stem cell line into which the phase separation marker (G3BP1) gene-specific fluorescent marker of the present invention was introduced, as disclosed in FIG. 3, had no abnormality in the expression of the pluripotency marker.

[0089]

[0090] Example 3. Verification of differentiation potential into three germ layers of a human pluripotent stem cell line into which a phase separation marker (G3BP1) gene-specific fluorescent marker has been introduced.

[0091] To verify the differentiation potential of the human induced pluripotent stem cell line CMC-hiPSC-009 (hereinafter referred to as CMC9_G3BP1-EGFP) into which a G3BP1 target fluorescent marker was introduced, embryoid body formation was induced and markers for each were analyzed.

[0092]

[0093] 1) Formation of the triploblast

[0094] Stem cells were seeded at 3×10 per well in an Ultra-low attachment 96-well plate. 3 After dividing into individual cells, they were cultured in a 37℃, 5% CO2 incubator for 3 days.

[0095] TeSR™-E6, which does not contain the differentiation inhibitor bFGF (basic fibroblast growth factor), was used as the culture medium for embryoid body formation. The culture medium was changed every two days, and the culture medium was carefully removed using a pipette and then new TeSR™-E6 was added. On the third day of culture, the morphological analysis of the embryoid bodies was performed through microscopic observation.

[0096] As a result, as disclosed in Figure 4A, CMC9_G3BP1-EGFP was confirmed to have morphologically formed a well-formed embryo.

[0097]

[0098] 2) Expression analysis of three-germ marker genes using qPCR

[0099] Embryos were harvested on the third day of culture, RNA was extracted using TRIzol (Invitrogen), and cDNA was synthesized using GoScript™ Reverse Transcriptase (Promega). qPCR was then performed using GoTaq®qPCR Master Mix (Promega) with 25 ng / ㎕ of cDNA as a template, and the mRNA expression levels of ectoderm markers (PAX6, Nestin), mesoderm markers (Desmin, α-SMA), and endoderm markers (SOX17, GATA4) were analyzed.

[0100] As a result, as shown in Fig. 4B, the expression of each marker was significantly increased compared to before differentiation, and thus it was confirmed that CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1 target fluorescent marker of the present invention was introduced, can normally differentiate into the three germ layers.

[0101]

[0102] 3) Expression analysis of three-germ layer marker proteins using immunofluorescence

[0103] On the third day of culture, embryonic bodies were harvested, washed three times with DPBS, and fixed for 20 minutes with 4% PFA fixative, followed by three washes with DPBS. Permeabilization was then achieved by treating with 0.1% TritonX-100 for 10 minutes at room temperature. Subsequently, the cells were washed three times with DPBS and blocked with 1% BSA for 1 hour at room temperature.

[0104] Primary antibodies (Nestin, #MAB5326; α-SMA, #ab5694; SOX17, #AF1924) were diluted 1:200, 1:200, and 1:20, respectively, in DPBS solution containing 2% BSA and 0.1% Triton X-100 and incubated overnight at 4°C. After washing three times with DPBS, secondary antibodies (Goat anti-Mouse IgG(H+L), Alexa Fluor™ 594, #A11032; Goat anti-Rabbit IgG(H+L), Alexa Fluor™ 594, #A11037; Donkey anti-Goat IgG(H+L), Alexa Fluor™ 647, #A21447) were diluted 1:1,000 in DPBS solution containing 2% BSA and 0.1% Triton X-100 and incubated overnight at room temperature. The cells were allowed to react for 3 hours. After washing three times with DPBS, nuclear counterstaining was performed by reacting with Hoechst 33342 diluted 1:1,000 in DPBS for 20 minutes at room temperature, and the expression of ectoderm markers (Nestin), mesoderm markers (α-SMA), and endoderm markers (SOX17) was analyzed using a confocal microscope.

[0105] As a result, as disclosed in Fig. 4C, it was confirmed that CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1 target fluorescent marker of the present invention was introduced, had normal expression of ectoderm, mesoderm, and endoderm markers.

[0106]

[0107] Example 4. Analysis of phase separation phenomenon (stress granule formation) following NaAsO2 treatment in human pluripotent stem cell lines into which a phase separation marker (G3BP1) gene-specific fluorescent marker has been introduced.

[0108] Stress granule formation in CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1 target fluorescent marker of the present invention was introduced, was confirmed by treatment with NaAsO2.

[0109] Cells were seeded at 3 × 10 per well on an 18-well μ-Slide coated with Matrigel. 3 After dividing into individual cells, the cells were cultured in a 37℃, 5% CO2 incubator for 3 days. The culture medium used was mTeSR-1, and the culture medium was replaced with fresh medium every 24 hours.

[0110] On the third day of culture, the stem cell culture medium was treated with 400 μM NaAsO2 diluted for 30 minutes, washed three times with DPBS, fixed for 20 minutes with a fixative, and washed three times with DPBS. The fixed cells were then observed using a confocal microscope to determine whether stress granules formed in the cytoplasm.

[0111] As a result, it was confirmed that fluorescently labeled G3BP1 was well expressed as disclosed in Fig. 5, and when NaAsO2 was treated, phase separation phenomenon, specifically stress granules were formed. Stress granules were also well formed in the CMC9_G3BP1-EGFP cells of the present invention, and this could be confirmed through EGFP.

[0112]

[0113] Example 5. Differentiation into cardiomyocytes from human pluripotent stem cells into which a phase separation marker (G3BP1) gene-specific fluorescent marker has been introduced.

[0114] We analyzed whether the fluorescent marker and cardiomyocyte-specific marker were normally expressed through differentiation into cardiomyocytes from CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1 target fluorescent marker of the present invention was introduced.

[0115]

[0116] 1) Differentiation into cardiomyocytes

[0117] Stem cells were seeded at 4 × 10 per well in a 4-well plate coated with Matrigel. 4After dividing into individual cells, the cells were cultured in a 37℃, 5% CO2 incubator for 4 days. The culture medium used was mTeSR-1, and the culture medium was replaced with fresh medium every 24 hours.

[0118] On the 4th day of culture, differentiation was initiated by replacing the culture medium with RPMI-1640 (Cat#11875093) containing Supplement B-27, minus insulin (Cat#A1895601) diluted 50-fold and CHIR99021 10 μM, Matrigel 1:200.

[0119] On the first day of differentiation, the culture medium was replaced with RPMI-1640 containing Supplement B-27 minus insulin diluted 50-fold.

[0120] On the third day of differentiation, the culture medium was replaced with a 50-fold dilution of Supplement B-27 minus insulin in RPMI-1640 and then prepared to contain 5 μM IWP4.

[0121] On the fifth day of differentiation, the culture medium was replaced with a 50-fold dilution of RPMI-1640 supplemented with Supplement B-27 minus insulin. Thereafter, the medium was replaced with the same medium every two days.

[0122]

[0123] 2) Analysis of expression of G3BP1 gene-specific fluorescent marker protein through fluorescence microscopy

[0124] As a result of observing cardiomyocytes on the 18th day of differentiation using a fluorescence microscope, as shown in Fig. 6A, it was confirmed that CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1 target fluorescent marker of the present invention was introduced, normally expressed the G3BP1 target fluorescent marker EGFP even after differentiation into cardiomyocytes.

[0125]

[0126] 3) Analysis of expression of cardiomyocyte-specific marker proteins using flow cytometry

[0127] On day 18 of differentiation, cardiomyocytes were harvested, washed twice with DPBS, fixed for 20 minutes in 4% PFA fixative, and washed twice with DPBS. Cells were then permeabilized with Perm buffer III (BD Biosciences) for 30 minutes at 4°C and washed twice with DPBS.

[0128] Antibody (cTnT, #565744) was diluted 1:50 in DPBS solution containing 2% FBS and 0.1% sodium azide, incubated at 4°C for 30 minutes, and then washed three times with DPBS solution containing 2% FBS and 0.1% sodium azide. The expression of the cardiomyocyte-specific marker (cTnT) was then analyzed using a flow cytometer.

[0129] As a result, as disclosed in Fig. 6B, it was confirmed that the cardiomyocytes derived from CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1 target fluorescent marker of the present invention was introduced, showed normal expression of cardiomyocyte-specific markers. In addition, it was confirmed that the G3BP1 target fluorescent marker, EGFP, was normally expressed even after differentiation into cardiomyocytes.

[0130]

[0131] Example 6. Differentiation of human pluripotent stem cells into lung cells by introducing a phase separation marker (G3BP1) gene-specific fluorescent marker.

[0132] We analyzed whether the fluorescent marker and lung-specific marker were normally expressed through differentiation into lung cells from CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1 target fluorescent marker of the present invention was introduced.

[0133]

[0134] 1) Differentiation into lung cells

[0135] Stem cells were seeded at 2×10 per well in a 4-well plate coated with Matrigel. 5After dividing into individual cells, the cells were cultured in a 37℃, 5% CO2 incubator for 3 days. The culture medium used was mTeSR-1, and the culture medium was replaced with fresh medium every 24 hours.

[0136] On the third day of culture, differentiation into endoderm cells was induced using the STEMdiff™ Definitive Endoderm Kit (STEMCELL technologies) according to the manufacturer's protocol.

[0137] On the second day of differentiation, endoderm cells were harvested using TrypLE and seeded at 2 × 10 per well in a Matrigel-coated 4-well plate. 5 Each cell was seeded and cultured in a 37°C, 5% CO2 incubator for 24 hours using anterior endoderm-1 culture medium. Anterior endoderm-1 culture medium was prepared by diluting Supplement B-27 (Cat#17504044) and Supplement N-2 (Cat#17502048) 50-fold in DMEM / F12 (Gibco) to obtain a concentration of 1% P / S, 10 μM SB431542, and 100 ng / mL Noggin.

[0138] On the third day of differentiation, the medium was replaced with anterior endoderm-2 medium and cultured for 24 hours in a 37°C, 5% CO2 incubator. Anterior endoderm-2 medium was prepared by diluting Supplement B-27 (Cat#17504044) and Supplement N-2 (Cat#17502048) 50-fold in DMEM / F12 (Gibco) to obtain a concentration of 1% P / S, 10 μM SB431542, and 1 μM IWP4.

[0139] On the 4th day of differentiation, the lung cell culture medium was replaced and cultured for 7 more days in a 37℃, 5% CO2 incubator, and fresh medium was replaced every 2 days. The lung cell culture medium was prepared by diluting Supplement B-27 (Cat#17504044) and Supplement N-2 (Cat#17502048) 50-fold in DMEM / F12 (Gibco) to make a concentration of 1% P / S, FGF10 10 ng / mL, KGF 10 ng / mL, BMP4 10 ng / mL, ATRA 50 nM, and CHIP99021 3 μM.

[0140]

[0141] 2) Analysis of lung-specific marker protein expression using immunofluorescence

[0142] On day 11 of differentiation, lung cells were washed three times with DPBS, fixed for 20 minutes with 4% PFA fixative, and then washed three times with DPBS. Permeabilization was then achieved by treating with 0.1% TritonX-100 for 10 minutes at room temperature. Subsequently, cells were washed three times with DPBS and blocked with 1% BSA for 1 hour at room temperature.

[0143] The primary antibody (NKX2.1, #ab227652) was diluted 1:50 in DPBS solution containing 2% BSA and 0.1% Triton X-100 and reacted overnight at 4℃, washed three times with DPBS, and then the secondary antibody (Goat anti-Rabbit IgG (H + L), Alexa Fluor™ 594, #A11037) was diluted 1:1,000 in DPBS solution containing 2% BSA and 0.1% Triton X-100 and reacted for 3 hours at room temperature. After washing three times with DPBS, Hoechst 33342 was diluted 1:1,000 in DPBS and reacted for 20 minutes at room temperature to perform nuclear counterstaining, and the expression of the lung-specific marker (NKX2.1) was analyzed using a fluorescence microscope.

[0144] As a result, as disclosed in Fig. 7A, it was confirmed that lung cells derived from CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1 target fluorescent marker of the present invention was introduced, normally expressed lung-specific markers. In addition, it was confirmed that the G3BP1 target fluorescent marker, EGFP, was normally expressed even after differentiation into lung cells.

[0145]

[0146] 3) Analysis of lung-specific marker protein expression using flow cytometry

[0147] On day 11 of differentiation, lung cells were harvested, washed twice with DPBS, fixed for 20 minutes in 4% PFA fixative, and washed twice with DPBS. Cells were then permeabilized with Perm buffer III (BD Biosciences) for 30 minutes at 4°C and washed twice with DPBS.

[0148] Primary antibodies (CPM, #014-27501; MUC1, #566590) were diluted 1:100 and 1:50 in DPBS solution containing 2% FBS and 0.1% sodium azide, respectively, and incubated at 4°C for 30 minutes, followed by washing three times with DPBS solution containing 2% FBS and 0.1% sodium azide. For CPM, secondary antibodies (Goat anti-Mouse IgG (H+L), Alexa Fluor™ 647, #A21236) were diluted 1:1,000 in DPBS solution containing 2% FBS and 0.1% sodium azide, and incubated at 4°C for 30 minutes. After washing three times with DPBS solution containing 2% FBS and 0.1% sodium azide, the expression of lung-specific markers (CPM, MUC1) was analyzed using a flow cytometer.

[0149] As a result, as disclosed in Fig. 7B, it was confirmed that lung cells derived from CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1 target fluorescent marker of the present invention was introduced, normally expressed lung-specific markers. In addition, it was confirmed that the G3BP1 target fluorescent marker, EGFP, was normally expressed even after differentiation into lung cells.

[0150]

[0151] Example 7. Analysis of differentiation into lung organoids derived from human pluripotent stem cell lines with a phase separation marker (G3BP1) gene-specific fluorescent marker and phase separation phenomenon (stress granule formation) following NaAsO2 treatment.

[0152] We analyzed whether lung-specific markers were normally expressed through differentiation into lung organoids from CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1-targeting fluorescent marker of the present invention was introduced. Furthermore, the formation of stress granules was analyzed by treatment with NaAsO2.

[0153]

[0154] 1) Differentiation into lung organoids

[0155] Differentiation into lung organoids was performed following the method performed in Example 6 above. As described in Example 6, after differentiating stem cells into lung cells, lung cells were harvested using TrypLE for 3D culture, and lung cells were mixed with 60% GFR Matrigel (Growth factor reduced Matrigel) diluted with lung organoid culture medium, and droplets were formed at 50 μl per well in a 24-well culture vessel. After that, the Matrigel was solidified for 30 minutes in a 37°C, 5% CO2 incubator, and 700 μl of lung organoid culture medium was added per well, and cultured in a 37°C, 5% CO2 incubator until harvested for lung organoid analysis. At this time, the lung organoid culture medium was prepared by diluting Supplement B-27 (Cat#17504044) and Supplement N-2 (Cat#17502048) 50-fold in DMEM / F12 (Gibco) to have a concentration of 1% P / S, FGF10 10 ng / mL, KGF 10 ng / mL, BMP4 10 ng / mL, ATRA 50 nM, and CHIP99021 3 μM, and the medium was replaced with new medium every 48 to 72 hours.

[0156] As a result, it was confirmed that the lung organoid derived from CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1 target fluorescent marker of the present invention was introduced, formed a 3D structure and grew normally, as disclosed in FIG. 8A.

[0157]

[0158] 2) Analysis of lung-specific marker protein expression using immunofluorescence

[0159] After 30 days of differentiation, lung organoids were harvested, washed three times with DPBS, fixed for 20 minutes in 4% PFA fixative, and washed three times with DPBS. Permeabilization was then achieved by treating with 0.1% TritonX-100 for 10 minutes at room temperature. Subsequently, the organoids were washed three times with DPBS and blocked with 1% BSA for 1 hour at room temperature.

[0160] The primary antibody (NKX2.1, #ab227652) was diluted 1:50 in DPBS solution containing 2% BSA and 0.1% Triton X-100 and reacted overnight at 4℃, washed three times with DPBS, and then the secondary antibody (Goat anti-Rabbit IgG (H + L), Alexa Fluor™ 594, #A11037) was diluted 1:1,000 in DPBS solution containing 2% BSA and 0.1% Triton X-100 and reacted for 3 hours at room temperature. After washing three times with DPBS, Hoechst 33342 was diluted 1:1,000 in DPBS and reacted for 20 minutes at room temperature to perform nuclear counterstaining, and the expression of the lung-specific marker (NKX2.1) was analyzed using a fluorescence microscope.

[0161] As a result, as disclosed in FIG. 8B, it was confirmed that the lung organoid derived from CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1 target fluorescent marker of the present invention was introduced, had normal expression of the lung-specific marker.

[0162]

[0163] 3) Analysis of phase separation (stress granule formation) in lung organoids following NaAsO2 treatment

[0164] Stress granule formation in lung organoids derived from CMC9_G3BP1-EGFP, a human stem cell line into which the G3BP1 target fluorescent marker of the present invention was introduced, was confirmed by treatment with NaAsO2.

[0165] The culture medium of cultured lung organoids was treated with 400 μM NaAsO2 for 1 hour, washed three times with DPBS, fixed for 20 minutes with a fixative, and washed three times with DPBS. The fixed organoids were then observed using a confocal microscope to determine whether stress granules formed in the cytoplasm.

[0166] As a result, as disclosed in Fig. 8C, it was confirmed that fluorescently labeled G3BP1 was well expressed, and when NaAsO2 was treated, a phase separation phenomenon, specifically stress granules, were formed. Stress granules were also well formed in the lung organoids derived from the CMC9_G3BP1-EGFP stem cell line of the present invention, and this could be confirmed through EGFP. In addition, it was confirmed that EGFP, a G3BP1 target fluorescent marker, was normally expressed even after differentiation into lung organoids.

Claims

1. Human pluripotent stem cells with a fluorescent reporter for toxicity assessment that detects phase separation phenomena in real time.

2. Human pluripotent stem cells with a fluorescent reporter for toxicity evaluation, characterized in that the human pluripotent stem cells in paragraph 1 are embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC).

3. Human pluripotent stem cells with a fluorescent reporter for toxicity evaluation, characterized in that the phase separation phenomenon in paragraph 1 is stress granule formation.

4. In the first paragraph, the human pluripotent stem cell into which the fluorescent reporter for toxicity evaluation has been introduced is characterized in that a fluorescent labeling factor has been introduced into the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system.

5. A human pluripotent stem cell having a fluorescent reporter for toxicity evaluation introduced therein, wherein the fluorescent reporter for toxicity evaluation in the first paragraph is any one selected from the group consisting of firefly luciferase, renilla luciferase, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), mCherry, and DsRed.

6. In the fourth paragraph, the human pluripotent stem cell into which the fluorescent reporter for toxicity evaluation has been introduced uses the CRISPR-Cas9 system to introduce firefly luciferase, renilla luciferase, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), A human pluripotent stem cell characterized by introducing a fluorescent reporter for toxicity assessment, wherein the fluorescent reporter is selected from the group consisting of mCherry and DsRed.

7. A cell differentiated from a human pluripotent stem cell according to any one of claims 1 to 6.

8. In paragraph 7, the cell is characterized in that it is a normal or abnormal cell of any one of a lung cell, a heart cell, a blood cell, a liver cell, a pancreatic cell, a brain cell, a small intestine cell, a large intestine cell, a stomach cell, a kidney cell, and a skin cell.

9. An organoid differentiated from a human pluripotent stem cell according to any one of claims 1 to 6.

10. In claim 9, the organoid is characterized in that it mimics a normal or abnormal tissue of any one of the small intestine, large intestine, liver, pancreas, kidney, brain, lung, heart, hair root, and skin.

11. A composition for toxicity evaluation comprising a human pluripotent stem cell according to any one of claims 1 to 6, or a cell or organoid differentiated therefrom.

12. A composition for toxicity evaluation in claim 11, characterized in that the toxicity evaluation is a toxicity evaluation caused by any one of pesticides; household chemical products or industrial chemicals; and biological hazards including bacteria or viruses.

13. A composition for toxicity evaluation in claim 12, characterized in that the household chemical product or industrial chemical is sodium azide.

14. A composition for screening for a toxic substance comprising a human pluripotent stem cell according to any one of claims 1 to 6, or a cell or organoid differentiated therefrom.

15. A composition for screening toxic substances, characterized in that in clause 14, the toxic substance is any one of pesticides; household chemical products or industrial chemicals; and biological hazards including bacteria or viruses.

16. A method for producing human pluripotent stem cells with a fluorescent reporter for toxicity evaluation, comprising the step of introducing a fluorescent label to the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system.

17. A method for producing human pluripotent stem cells with a fluorescent reporter for toxicity evaluation, characterized in that the method comprises the steps of: transfecting stem cells with a CRISPR-Cas9-sgRNA vector containing sgRNA; and a gene insertion (Knock-in) vector for introducing a fluorescent marker into the G3BP1 gene; and introducing a fluorescent marker into the 3'-end of the G3BP1 gene.

18. A method for producing human pluripotent stem cells, wherein the sgRNA in claim 17 is characterized in that it consists of a base sequence of sequence number 1. 19.1) A step of treating a test substance for toxicity evaluation to a human pluripotent stem cell, a cell or organoid differentiated therefrom, according to any one of clauses 1 to 6; and 2) A method for screening a toxic substance, comprising: a step of predicting a toxic substance by confirming a real-time phase separation phenomenon after the above step 1).

20. A method for screening a toxic substance, characterized in that in claim 19, the cell differentiated from the human pluripotent stem cell of step 1) is any one of a normal or abnormal cell among a lung cell, a heart cell, a blood cell, a liver cell, a pancreatic cell, a brain cell, a small intestine cell, a colon cell, a stomach cell, a kidney cell, and a skin cell.

21. A method for screening a toxic substance in claim 19, wherein the organoid of step 1) is an organoid that mimics a normal or abnormal tissue of any one of the small intestine, large intestine, liver, pancreas, kidney, brain, lung, heart, hair root, and skin.

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