Human tissue stem cells and their uses

Abstract

To provide a technique of performing gene locus-specific gene manipulation on stem cells such as cancer stem cells.SOLUTION: Provided herein are a human tissue stem cell, a method of producing a human tissue stem cell, and a method of testing the effectiveness of an anticancer drug against a human cancer stem cell and a human cancer cell. The human tissue stem cell has a gene construct including IRES-target gene introduced into a gene locus of a stem cell marker gene. The stem cell marker gene is a LGR5 gene. The gene construct is stably introduced into exon 18 of the LGR5 gene locus. It has both capabilities of self-replication and differentiation for a long period of time.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to human tissue stem cells and uses thereof. More specifically, the present invention relates to human tissue stem cells, a method for introducing a gene of interest into the locus of a stem cell marker gene of human tissue stem cells, a method for testing whether a human tissue stem cell is a stem cell, a method for screening an anticancer agent effective against cancer stem cells, and a method for testing the effectiveness of an anticancer agent against cancer stem cells and cancer cells. This application claims priority based on Japanese Patent Application No. 2017-063171 filed in Japan on March 28, 2017, the contents of which are incorporated herein by reference. [Background technology]

[0002] The cancer stem cell theory focuses on a subpopulation of cancer cells that support tumor growth and self-replicate (see, for example, Non-Patent Document 1). According to the cancer stem cell theory, it is believed that a small number of cancer stem cells are present in tumor tissue from the early stage of carcinogenesis, and that tumor tissue is formed by the repeated self-replication and differentiation of cancer stem cells. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Kreso A. and Dick JE, Evolution of the cancer stem cell model. Cell Stem Cell, 4 (14), 275-291, 2014. Summary of the Invention [Problem to be solved by the invention]

[0004] However, no technology has been established for performing locus-specific genetic manipulation of stem cells such as cancer stem cells. For this reason, it has been difficult to perform lineage analysis of tissue stem cells, particularly human ones, and to confirm that a single cell has the ability to self-replicate and differentiate. Therefore, an object of the present invention is to provide a technology for performing locus-specific genetic manipulation of tissue stem cells. [Means for solving the problem]

[0005] The present invention includes the following aspects. [1] Human tissue stem cells with a target gene introduced into the locus of a stem cell marker gene. [2] The human tissue stem cell described in [1], wherein the stem cell marker gene is the Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) gene. [3] The human tissue stem cell described in [2], in which a gene of interest has been introduced into exon 18 of the Lgr5 locus. [4] The human tissue stem cell according to [1] or [2], wherein the target gene is a reporter gene. [5] The human tissue stem cell described in any one of [1] to [4], wherein the target gene is a first reporter gene, and a second reporter gene driven by a promoter for forced expression is further introduced. [6] The human tissue stem cell described in any one of [1] to [5], which is a stem cell derived from cancer tissue. [7] A method for introducing a gene of interest into a locus of a stem cell marker gene of a human tissue stem cell, the method comprising a step of introducing the gene of interest into the locus of the stem cell marker gene of a cultured organoid containing human tissue stem cells by genome editing. [8] The method according to [7], wherein the step of introducing the target gene is performed by electroporation, and further comprises a step of culturing the cultured organoid at 30°C for 2 days after introducing the target gene. [9] The method according to [7] or [8], wherein the locus of the stem cell marker gene is the Lgr5 locus.

[10] The method described in [9], wherein the target gene is introduced into exon 18 of the Lgr5 locus.

[11] The method according to any one of [7] to

[10] , wherein the target gene is a gene encoding a lineage marking-inducing protein.

[12] A method for testing whether a human tissue stem cell produced by the method described in

[11] is a stem cell, comprising a step of detecting self-renewal and differentiation of the human tissue stem cell by cell lineage analysis of the human tissue stem cell, wherein detection of both self-renewal and differentiation of the human tissue stem cell indicates that the human tissue stem cell is a stem cell.

[13] The method according to any one of [7] to

[12] , wherein the human tissue stem cells are stem cells derived from cancer tissue.

[14] A method for screening for an anticancer drug effective against cancer stem cells, comprising the steps of culturing cancer stem cells into which a reporter gene driven by the promoter of a cancer stem cell marker gene has been introduced in the presence of a test substance, and measuring the expression level of the reporter gene, wherein a decrease in the expression level of the reporter gene indicates that the test substance is an anticancer drug effective against cancer stem cells.

[15] A method for testing the effectiveness of an anticancer agent against cancer stem cells and cancer cells, comprising the steps of culturing cancer stem cells into which a first reporter gene driven by a promoter of a cancer stem cell marker gene and a second reporter gene driven by a promoter for forced expression have been introduced in the presence of the anticancer agent, and measuring the expression levels of the first and second reporter genes, wherein a decrease in the expression level of the first reporter gene indicates that the anticancer agent is an effective anticancer agent against cancer stem cells, and a decrease in the expression level of the second reporter gene indicates that the anticancer agent is an effective anticancer agent against cancer stem cells and cancer cells.

[0006] The present invention can also be said to include the following aspects. [P1] Tissue stem cells into which a target gene has been introduced at a target locus. [P2] The tissue stem cell according to [P1], wherein the target gene locus is the locus of a stem cell marker gene. [P3] The tissue stem cell described in [P2], wherein the stem cell marker gene is the Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) gene. [P4] The tissue stem cell according to [P3], wherein the target gene is a reporter gene. [P5] The tissue stem cell according to any one of [P1] to [P4], wherein the target gene is a first reporter gene, and a second reporter gene driven by a promoter for forced expression is further introduced. [P6] The tissue stem cell according to any one of [P1] to [P5], which is a stem cell derived from cancer tissue. [P7] A method for introducing a target gene into a target locus of a tissue stem cell, the method comprising a step of introducing the target gene into the target locus of a cultured organoid containing tissue stem cells. [P8] The method according to [P7], wherein the step of introducing the target gene is performed by electroporation, and further comprises a step of culturing the cultured organoid at 30°C for 2 days after introducing the target gene. [P9] The method according to [7] or [8], wherein the target locus is a stem cell marker locus. [P10] The method according to [P9], wherein the target gene is a gene encoding a cell lineage marking-inducing protein. [P11] [P10] A method for testing whether or not a tissue stem cell produced by the method described therein is a stem cell, the method comprising a step of detecting self-renewal and differentiation of the tissue stem cell by cell lineage analysis of the tissue stem cell, wherein detection of both self-renewal and differentiation of the tissue stem cell indicates that the tissue stem cell is a stem cell. [P12] The method according to any one of [P7] to [P11], wherein the tissue stem cells are stem cells derived from cancer tissue. [P13] A method for screening for anticancer drugs effective against cancer stem cells, comprising the steps of culturing cancer stem cells into which a reporter gene driven by the promoter of a cancer stem cell marker gene has been introduced in the presence of a test substance, and measuring the expression level of the reporter gene, wherein a decrease in the expression level of the reporter gene indicates that the test substance is an anticancer drug effective against cancer stem cells. [P14] A method for testing the effectiveness of an anticancer drug against cancer stem cells and cancer cells, comprising the steps of culturing cancer stem cells into which a first reporter gene driven by a promoter of a cancer stem cell marker gene and a second reporter gene driven by a promoter for forced expression have been introduced in the presence of the anticancer drug, and measuring the expression levels of the first and second reporter genes, wherein a decrease in the expression level of the first reporter gene indicates that the anticancer drug is an effective anticancer drug against cancer stem cells, and a decrease in the expression level of the second reporter gene indicates that the anticancer drug is an effective anticancer drug against cancer stem cells and cancer cells. Effect of the Invention

[0007] According to the present invention, a technique for performing locus-specific genetic manipulation on tissue stem cells can be provided. [Brief description of the drawings]

[0008] [Figure 1] (a) is a schematic diagram of the human LGR5 locus. (b) is a schematic diagram showing the structure of the targeting construct prepared in Experimental Example 1. (c) is a schematic diagram showing the structure of the LGR5 locus of the organoid prepared in Experimental Example 1. [Diagram 2] (a) to (c) are fluorescence micrographs of tissue sections of tumor tissue derived from the organoids transplanted into mice in Experimental Example 2. (d) to (f) are fluorescence micrographs of sections of patient-derived colon cancer tissue from which organoid clones were established in Experimental Example 2. [Diagram 3] 1(a) to (c) are diagrams illustrating labeling with cell lineage markers. [Figure 4] FIG. 13 is a diagram illustrating the structure of the LGR5 gene locus in the organoid prepared in Experimental Example 4 and the behavior of the drug-inducible suicide gene. [Diagram 5] 1(a) is a fluorescent micrograph showing the results of observing a tumor tissue section from a control mouse in Experimental Example 4. FIG. 1(b) is a fluorescent micrograph showing the results of observing a tumor tissue section from a mouse administered AP20187 in Experimental Example 4. [Figure 6] 1 is a graph showing the results of measuring the tumor size over time in AP20187-administered mice (Dimerizer) and control mice (Vehicle) in Experimental Example 4. [Figure 7] 13(a) and (b) are graphs showing the results of measuring the size of tumor tissue derived from organoids over time in Experimental Example 5. [Figure 8] 1 shows microscopic images of organoids showing representative results of Experimental Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] [Tissue stem cells] In one embodiment, the present invention provides a tissue stem cell having a target gene introduced into a target locus.

[0010] In this embodiment, the tissue stem cells may be human cells or non-human animal cells. Stem cells are cells that have the ability to self-replicate and differentiate. Tissue stem cells are also called somatic stem cells and are stem cells present in tissues, and are different from embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). In this specification, tissue stem cells include cancer stem cells. Furthermore, the tissue stem cells of this embodiment include cells that can be dedifferentiated into stem cells due to cellular plasticity.

[0011] Conventionally, it has been difficult to carry out locus-specific genetic manipulation of tissue stem cells. In contrast, as described later in the Examples, the inventors have revealed that by carrying out locus-specific genetic manipulation of cultured organoids containing tissue stem cells, tissue stem cells having a target gene introduced into a target locus can be produced.

[0012] In the tissue stem cells of this embodiment, the locus of interest may be any locus of interest, for example, the locus of a stem cell marker gene. More specifically, the tissue stem cells of this embodiment are cancer stem cells, and the locus of interest may be the locus of a cancer stem cell marker gene. Examples of cancer stem cell marker genes include, but are not limited to, LGR5, SPARC Related Modular Calcium Binding 2 (SMOC2), Repulsive Guidance Molecule Family Member B (RGMB), AXIN2, Olfactomedin 4 (OLFM4), Cell Division Cycle Associated 7 (CDCA7), Leucine Rich Repeats And Immunoglobulin Like Domains 1 (LRIG1), Ring Finger Protein 43 (RNF43), Achaete-Scute Family BHLH Transcription Factor 2 (ASCL2), and the like.

[0013] In addition, the cancer from which the cancer stem cells originate is not particularly limited, and may be, for example, cancer stem cells of colon cancer, gastric cancer, or pancreatic cancer. The inventors have confirmed that the LGR5 gene can be used as a cancer stem cell marker in these cancer stem cells.

[0014] The inventors have also demonstrated that by performing locus-specific genetic manipulation on cultured organoids derived from normal cells, it is possible to introduce a target gene into a target locus in tissue stem cells of normal cells.

[0015] Here, whether a cultured organoid is derived from a normal cell or a cancer cell can be determined by the requirement of growth factors necessary for organoid culture, confirmation of gene mutations by sequencing, confirmation of chromosomal abnormalities, etc.

[0016] Furthermore, in the stem cells of this embodiment, the target gene may be any gene. For example, the target gene may be a reporter gene. Furthermore, the reporter gene may be introduced into a region other than the first exon of the gene located at the target gene locus. For example, the reporter gene may be introduced into the 18th exon of the LGR5 gene. Here, the reporter gene is not particularly limited, and examples thereof include various fluorescent proteins, enzymes, etc.

[0017] In locus-specific gene manipulation by genome editing and the like, a gene of interest is often introduced into the first exon of a target gene. The inventors first attempted to introduce a reporter gene into the first exon of the LGR5 gene of colon cancer stem cells. However, this gene introduction did not go well. In contrast, as described later in the Examples, when a reporter gene was introduced into the 18th exon of the LG5 gene, the desired gene introduction could be efficiently performed.

[0018] The gene of interest to be introduced into the locus of interest may not have a promoter sequence, i.e., the gene of interest may be configured to be expressed by the promoter of the locus of interest.

[0019] For example, if an Internal R i By linking an IRES or a ribosomal skip site such as T2A or P2A, the target gene can be expressed by the promoter of the target gene locus.

[0020] The tissue stem cell of this embodiment may be a cancer stem cell into which a second reporter gene driven by a promoter for forced expression has been introduced in addition to a first reporter gene driven by a promoter of the marker gene introduced into the locus of the cancer stem cell marker gene. Here, the second reporter gene does not have to be introduced in a locus-specific manner. In addition, as the promoter for forced expression, a promoter that is usually used for forced expression of genes in animal cells can be used, and examples thereof include, but are not limited to, the CMV promoter and the EF1α promoter.

[0021] Here, the first and second reporter genes are preferably distinguishable from each other. For example, the first and second reporter genes may be genes encoding fluorescent proteins that emit fluorescence with different wavelengths.

[0022] Here, the first reporter gene is expressed when the cancer stem cell marker gene is expressed, that is, when the cell maintains cancer stemness. Meanwhile, the second reporter gene is expressed regardless of the expression of the cancer stem cell marker gene, that is, when the cancer stem cell maintains cancer stemness or when it loses cancer stemness. Therefore, the expression of the first reporter gene indicates the presence of cancer stem cells, and the expression of the second reporter gene indicates both the presence of cancer stem cells or the presence of cancer cells. Here, cancer cells refer to cancer cells that do not express the cancer stem cell marker gene.

[0023] As described later in the Examples, such cancer stem cells are cultured in the presence of a test substance, and the expression levels of the first and second reporter genes are measured, respectively, to separately evaluate the effects on cancer stem cells and cancer cells. Therefore, such cancer stem cells can be used for screening of anticancer agents and the like that are effective against cancer stem cells.

[0024] [Method for introducing a target gene into a target locus in tissue stem cells] In one embodiment, the present invention provides a method for introducing a target gene into the target locus of tissue stem cell, comprising the step of introducing the target gene into the target locus of the cultured organoid that comprises tissue stem cell.The method of this embodiment can be said to be the method comprising the step of introducing a target gene into the target locus of the cell that constitutes cultured organoid.

[0025] In the method of this embodiment, the stem cells are similar to those described above, and examples thereof include cancer stem cells and somatic stem cells.

[0026] Conventionally, it has been difficult to carry out genetic manipulation on stem cells such as cancer stem cells and tissue stem cells. In particular, it has been difficult to carry out locus-specific genetic manipulation on tissue stem cells. In contrast, as described later in the Examples, the inventors have revealed that by modifying the genes of cultured organoids containing tissue stem cells, cultured organoids into which a target gene has been introduced can be produced.

[0027] Furthermore, as described later in the Examples, the method of this embodiment also allows locus-specific genetic manipulation of tissue stem cells, thereby producing tissue stem cells in which a target gene has been introduced into a target locus.

[0028] In this specification, organoid means organ-like tissue obtained by three-dimensional culture of cells.In this specification, cell mass is also included in organoid.The method of three-dimensional culture is not particularly limited, and for example, the method using Matrigel, the method using collagen, the method using laminin, etc. can be mentioned.

[0029] In the method of the present embodiment, the cultured organoid is not particularly limited as long as it is an organoid containing tissue stem cells, and examples thereof include organoids of epithelial cells, organoids of epithelial tumor cells, etc. Examples of epithelial cells include digestive epithelial cells such as stomach, small intestine, large intestine, bile duct, and pancreatic duct epithelium, and epithelial cells of tissues other than the digestive organs such as mammary gland and prostate.

[0030] As the method for culturing organoid, known culturing methods can be used.In particular, in the method for culturing stem cells derived from cancer tissue, the culturing method optimized for each cancer tissue can be selected (for example, see Fujii M., et al., Nature Protocols, Vol.10, 1474-1485, 2015; Mihara E., et al., eLIFE, Vol.5, E11621, 2016; Fujii M., et al., Cell Stem Cell, Vol.18, 827-838, 2016, etc.).

[0031] In the method of the present embodiment, the step of introducing a target gene into cultured organoids can be performed by a known gene introduction method. In particular, as shown in the examples, it is preferable to perform the step by electroporation. Furthermore, it is preferable to include a step of culturing the cultured organoids at 30°C for 2 days after electroporation. The inventors have revealed that by including the above steps, the efficiency of gene introduction into organoids can be significantly improved.

[0032] [Method for testing whether tissue stem cells are stem cells] In one embodiment, the present invention provides a method for testing whether a tissue stem cell labeled with a lineage marker driven by a promoter of a gene of interest is a stem cell, comprising detecting self-renewal and differentiation of the tissue stem cell by lineage analysis of the tissue stem cell, and detecting both self-renewal and differentiation of the tissue stem cell indicates that the tissue stem cell is a stem cell. The lineage marker will be described later.

[0033] The method of the present embodiment can also be said to be a method for proving that a cell believed to be a tissue stem cell is a stem cell. The method of the present embodiment can also be said to be a method for testing whether a target gene is a stem cell marker. That is, the method of the present embodiment is a method for testing whether a target gene is a stem cell marker, comprising a step of culturing a cell labeled with a cell lineage marker driven by a promoter of the target gene, and a step of detecting self-renewal and differentiation of the cell by cell lineage analysis of the cell, and the detection of both self-renewal and differentiation of the cell can also be said to be a method for indicating that the target gene is a stem cell marker.

[0034] Stem cells are cells that have the ability to self-renew and differentiate. Therefore, to prove that a target cell is a stem cell, it is necessary to show that a single target cell performs both self-renewal and differentiation. Alternatively, to prove that a target gene is a stem cell marker, it is necessary to show that a single cell expressing the target gene performs both self-renewal and differentiation.

[0035] In order to demonstrate that a single cell can perform both self-renewal and differentiation, it is necessary to demonstrate that cells derived from a single cell can perform both self-renewal and differentiation by cell lineage analysis, which tracks and observes cells derived from that single cell. Cell lineage analysis is an analytical method that identifies and tracks cells that are descendants of a single cell of interest, even if that cell undergoes repeated cell division or undergoes changes in properties due to differentiation.

[0036] As used herein, a cell lineage marker refers to a marker that can identify a cell as a descendant of a single subject cell, even if that cell undergoes repeated cell division or undergoes changes in properties due to differentiation.

[0037] In the method of this embodiment, cells expressing a gene of interest are labeled with a lineage marker driven by the promoter of the gene of interest. As a result, the cells expressing the gene of interest and their progeny can be identified and observed. If the labeled cells are then confirmed to have undergone both self-renewal and differentiation, the gene of interest can be proven to be a stem cell marker. The lineage marker preferably drives the promoter of the gene of interest at the locus of interest.

[0038] The cell lineage marker is not particularly limited as long as it can label cells so as to trace their descendants, and can be, for example, a combination of a cell lineage labeling-inducing protein expressed by a promoter of a target gene and a cell lineage labeling construct separately introduced into a cell, in which the cell lineage labeling-inducing protein acts on the cell lineage labeling construct to label the cell lineage-wise.

[0039] More specific examples of cell lineage markers include those that utilize the Cre-LoxP system and variants of the Cre-loxP system such as the VCre-VLoxP system and the SCre-SloxP system; those that utilize the Flp-Frt system using recombinase derived from budding yeast; and combinations of these.

[0040] Examples of the cell lineage marking induction protein include Cre recombinase, VCre recombinase, SCre recombinase, and Flp recombinase.

[0041] The cell lineage labeling construct may be, for example, one that can perform cell lineage labeling using the cell lineage labeling induction protein used. For example, when the cell lineage labeling induction protein is Cre recombinase, a construct that has a loxP sequence and can be detected when genetic recombination occurs may be used. The cell lineage labeling construct may be, for example, configured to express a fluorescent protein when genetic recombination occurs.

[0042] In such a system, when a gene of interest is expressed, genetic recombination occurs and the cell expresses, for example, a fluorescent protein, which continues to express the fluorescent protein even after division, allowing for cell lineage analysis.

[0043] In the method of this embodiment, the tissue stem cells transfected with the lineage tagging protein driven by the promoter of the target gene can be prepared by the above-mentioned method. Meanwhile, the lineage tagging construct does not have to be transfected in a locus-specific manner.

[0044] Conventionally, it has been difficult to perform locus-specific genetic manipulation on tissue stem cells. Therefore, it has been difficult to introduce a cell lineage labeling induction protein that operates on the promoter of a target gene into tissue stem cells. However, as described above, the inventors have revealed that it is possible to perform locus-specific genetic manipulation on tissue stem cells by a method of gene introduction into cultured organoids.

[0045] As a result, it became possible to introduce a lineage marking-inducing protein that operates on the promoter of a target gene into tissue stem cells, and separately introduce a lineage marking construct to label the tissue stem cells with a lineage marker and test whether the target gene is a stem cell marker.

[0046] [Method for screening anticancer drugs effective against cancer stem cells] In one embodiment, the present invention provides a method for screening anticancer agents effective against cancer stem cells, comprising the steps of culturing cancer stem cells into which a reporter gene driven by the promoter of a cancer stem cell marker gene has been introduced in the presence of a test substance, and measuring the expression level of the reporter gene, wherein a decrease in the expression level of the reporter gene indicates that the test substance is an anticancer agent effective against cancer stem cells.

[0047] In the screening method of this embodiment, cancer stem cell can be cultured in the form of organoid containing cancer stem cell.In addition, cancer stem cell is preferably proven to be cancer stem cell by the above-mentioned method.That is, cancer stem cell marker gene is preferably proven to be cancer stem cell marker by the above-mentioned method.

[0048] Furthermore, the reporter gene is not particularly limited, and examples thereof include various fluorescent proteins, enzymes, etc. It is preferable that the reporter gene be driven by a promoter in the gene locus of the cancer stem cell marker gene.

[0049] As described later in the Examples, the screening method of the present embodiment can measure the effect of the test substance on cancer stem cells. As a result, it is possible to screen for an anticancer drug that is effective against cancer stem cells. The test substance is not particularly limited, and may be, for example, a compound library or a library of existing drugs including antibody drugs.

[0050] In another embodiment, the present invention provides a method for testing the effectiveness of an anticancer agent against cancer stem cells and cancer cells, comprising the steps of culturing cancer stem cells into which a first reporter gene driven by a promoter of a cancer stem cell marker gene and a second reporter gene driven by a promoter for forced expression have been introduced in the presence of the anticancer agent, and measuring the expression levels of the first and second reporter genes, wherein a decrease in the expression level of the first reporter gene indicates that the anticancer agent is an effective anticancer agent against cancer stem cells, and a decrease in the expression level of the second reporter gene indicates that the anticancer agent is an effective anticancer agent against cancer stem cells and cancer cells.

[0051] The method of the present embodiment can also be said to be a method for screening anticancer drugs. As the promoter for forced expression, a promoter that is usually used for forced expression of genes in animal cells can be used, and examples thereof include, but are not limited to, CMV promoter, EF1α promoter, etc.

[0052] In the method of this embodiment, the first reporter gene is preferably driven by a promoter in the gene locus of a cancer stem cell marker gene. In addition, the first reporter gene and the second reporter gene are preferably distinguishable from each other. For example, the first reporter gene and the second reporter gene may be genes encoding fluorescent proteins that emit fluorescence with different wavelengths.

[0053] As described later in the Examples, the method of this embodiment can evaluate the effects of a target anticancer drug on cancer stem cells and cancer cells, respectively. Therefore, the method of this embodiment can be used for screening anticancer drugs and the like that are effective against cancer stem cells. EXAMPLES

[0054] The present invention will now be described in more detail with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[0055] [Experimental Example 1] (Introduction of a reporter gene into the LGR5 locus of colon cancer stem cells) Creating a Construct A CRISPR-Cas9 plasmid targeting the 18th exon of the LGR5 gene was prepared by a standard method. Figure 1(a) is a schematic diagram of the human LGR5 locus. In addition, a targeting construct was prepared to introduce a reporter gene into the 18th exon of the LGR5 gene. A gene encoding green fluorescent protein (GFP) was used as the reporter gene. Figure 1(b) is a schematic diagram showing the structure of the targeting construct prepared.

[0056] The targeting construct was prepared by ligating a 5' arm and a 3' arm, each having a length of 1 kbp, to an IRES-GFP-loxP-EF1-RFP-T2A-Puro-loxP plasmid (type "HR180PA-1", System Biosciences).

[0057] Organoid culture The experiment was performed with approval from the Ethics Committee of Keio University School of Medicine. Colon cancer tissues removed from patients were cut into small pieces and washed at least 10 times with ice-cold phosphate buffered saline (PBS). Liberase (type "TH", Roche Life Sciences) was then added and digested at 37°C for 60 minutes with vigorous pipetting every 15 minutes. The remaining fragments were then further digested with enzyme (type "TrypLE Express", Invitrogen) at 37°C for 20 minutes.

[0058] The supernatant was then collected and centrifuged at 200×g for 3 minutes at 4° C. The precipitate was then suspended in Matrigel (BD Biosciences) and dispensed into a 24-well plate at 25 μL / well. After the Matrigel was polymerized, a medium was added.

[0059] The medium used was Dulbecco's modified Eagle / F12 medium supplemented with penicillin, streptomycin, 10 mM HEPES, 2 mM GlutaMAX (Thermo Fisher Scientific), 1xB27 (Life Technologies), 10 nM Gastrin I (Sigma), and 1 mM N-acetylcysteine ​​(Wako Pure Chemical Industries, Ltd.). In some cases, 50 ng / mL mouse recombinant EGF, 100 ng / mL mouse recombinant Noggin, and 500 nM A83-01 (Tocris) were added to the medium.

[0060] The inventors have previously demonstrated that colon cancer organoids can be cultured for a long period of time using the above-mentioned culture method, and that the colon cancer organoids contain colon cancer stem cells.

[0061] Genetic Introduction Genes were introduced into the above-mentioned cultured organoids by electroporation. After gene introduction, the cultured organoids were cultured at 30°C for 2 days. Then, 3 days after gene introduction, selective culture was performed in the presence of 2 μg / mL puromycin for 3 days.

[0062] We then selected and individually expanded drug-resistant organoid clones, and extracted genomic DNA from each organoid clone to confirm that the desired gene had been introduced by PCR and Southern blotting.

[0063] The RFP-T2A-Puro selection cassette, flanked by loxP sequences, was then removed by transient infection with an adenovirus expressing Cre recombinase. RFP-negative organoids were then selected and cloned. PCR was then used to confirm that the RFP-T2A-Puro selection cassette had been removed.

[0064] By the above method, three clones of organoids, named CRC7, CRC12, and CRC28, were obtained. Figure 1(c) is a schematic diagram showing the structure of the LGR5 locus of the obtained organoids. Hereinafter, these organoids may be referred to as "LGR5-GFP organoids."

[0065] [Experimental Example 2] (Identifying the stem cell hierarchy) 《Xenotransplantation of Organoids》 Animal experiments were performed with the approval of the Keio University School of Medicine Animal Experiment Committee. The three LGR5-GFP organoid clones prepared in Experimental Example 1 were xenografted under the renal capsule of immunodeficient mice. NOD / Shi-scid IL-2Rγnull (NOG) mice (7-12 weeks old, male) were used as immunodeficient mice.

[0066] Detection of cancer stem cells and differentiated cancer cells Tumors were excised from each mouse and immediately fixed in 4% paraformaldehyde, then frozen sections were prepared. GFP fluorescence, which indicates the presence of cancer stem cells, was then detected in the tissue sections. Keratin 20 (KRT20), a differentiation marker, was also detected by immunohistochemistry.

[0067] Figures 2(a) to (c) are fluorescence microscopy images of tissue sections of tumor tissue derived from transplanted organoids. In Figure 2, "Xenograft" indicates tumor tissue derived from xenografted organoids, and "Patient" indicates tumor tissue derived from a patient, as described below. The scale bar indicates 100 μm. Figure 2(a) shows the results of tumor tissue derived from LGR5-GFP organoid clone CRC7, Figure 2(b) shows the results of tumor tissue derived from clone CRC12, and Figure 2(c) shows the results of tumor tissue derived from clone CRC28.

[0068] The results showed that GFP-positive cells (cancer stem cells) were present in the outermost region of the tumor, while KRT20 (differentiated cells) were present inside the tumor.

[0069] In addition, Figures 2(d) to (f) are fluorescent micrographs showing the results of detecting LGR5 mRNA by in situ hybridization and KRT20 by immunostaining in sections of patient-derived colon cancer tissue from which each organoid clone was established.

[0070] Figure 2(d) shows the results for the patient's tumor tissue used to establish clone CRC7, Figure 2(e) shows the results for the patient's tumor tissue used to establish clone CRC12, and Figure 2(f) shows the results for the patient's tumor tissue used to establish clone CRC28.

[0071] As a result, it was revealed that in the patient's tumor tissue, LGR5-expressing cells (cancer stem cells) were present in the outermost region of the tumor, while KRT20 (differentiated cells) were present inside the tumor.

[0072] These results confirmed that the stem cell hierarchy in the patient's tumor tissue was recapitulated in the organoid-derived tumor tissue.

[0073] [Experimental Example 3] (Proof that LGR5 gene-expressing cells are cancer stem cells) We labeled cancer stem cells with lineage markers and performed lineage analysis to examine whether a single cell can undergo both self-renewal and differentiation.

[0074] <<Labeling with lineage markers>> First, cancer stem cells were labeled with a cell lineage marker driven by the LGR5 gene promoter. Figures 3(a) to (c) are diagrams explaining the labeling with the cell lineage marker.

[0075] First, an IRES-CreER construct was introduced into the LGR5 locus of a cultured organoid derived from colon cancer by the same method as in Experimental Example 1. The CreER protein is a recombinase that is normally present in the cytoplasm, but is translocated into the nucleus upon binding to tamoxifen and causes recombination at the loxP sequence. Figure 3(a) is a schematic diagram of the completed LGR5 locus.

[0076] In addition, a reporter cassette was further introduced into the organoid into which IRES-CreER was introduced by the same method as in Experimental Example 1. As the reporter cassette, a rainbow cassette excised from a CMV-Brainbow-2.1R plasmid (type number "#18723", Addgene Inc.) was used. Figure 3(b) is a schematic diagram of the rainbow cassette.

[0077] Here, we will explain the organoids labeled with the cell lineage markers shown in Figures 3(a) and (b). Cancer stem cells present in the organoids labeled with the above cell lineage markers express the LGR5 gene, and therefore express the CreER protein. In the absence of tamoxifen, CreER is present in the cytoplasm, so recombination of the rainbow cassette does not occur. As a result, nuclear GFP (nGFP) is expressed, and GFP fluorescence is observed in the nucleus.

[0078] When tamoxifen is administered to the cells, CreER translocates into the nucleus, and random recombination occurs in the multiple loxP sequences present in the rainbow cassette. As a result, for example, the gene sequence shown in Figure 3(c) is formed. As a result, the cells express either yellow fluorescent protein (YFP), red fluorescent protein (RFP), or cyan fluorescent protein (mCFP), depending on the case.

[0079] Once a cell has undergone recombination to express one of the fluorescent proteins, the cell and its descendants continue to express the same fluorescent protein. This allows cells derived from a single cell to be tracked and analyzed. Hereinafter, organoids labeled with the above cell lineage markers may be referred to as "LGR5-CreER / Rainbow organoids."

[0080] <Lineage analysis of cancer stem cells> Cancer stem cells express the LGR5 gene, and therefore, the cancer stem cells contained in the LGR5-CreER / Rainbow organoid express CreER and, in the presence of tamoxifen, change from cells that emit nGFP fluorescence to cells that emit YFP, RFP, and mCFP fluorescence.

[0081] Using this system, we performed cell lineage analysis of cancer stem cells. Specifically, first, LGR5-CreER / Rainbow organoids were xenografted under the renal capsule of NOG mice (7-12 weeks old, male) in the same manner as in Experimental Example 2. Then, tamoxifen was administered to the mice one month after the transplantation. Then, tumors were excised from the mice 3 and 28 days after the administration of tamoxifen, and immediately fixed with 4% paraformaldehyde to prepare frozen tissue sections.

[0082] Next, the behavior of cancer stem cells was observed by performing fluorescent observation of the prepared tissue sections, in situ hybridization for LGR5 mRNA, and immunostaining for KRT20.

[0083] The results showed that cancer stem cells were present in the outermost region of tumor tissue immediately after tamoxifen administration, and that tumor tissue was subsequently generated continuously by lineage-tagged cell clones.

[0084] In addition, it was revealed that the cells that initially expressed the LGR5 gene formed a clonal structure containing both LGR5-positive and LGR5-negative cells, and that the cells that initially expressed the LGR5 gene did not express KRT20, but differentiated into KRT20-positive cells one week after tamoxifen administration.

[0085] These results demonstrated that LGR5-positive cells are cancer stem cells with long-term self-renewal and differentiation capabilities in humans, and that the LGR5 gene is a cancer stem cell marker in humans.

[0086] [Experimental Example 4] (Investigating the effectiveness of cancer treatments that target cancer stem cells) We transduced a drug-induced suicide gene into cancer stem cells and investigated the effectiveness of cancer therapy targeting cancer stem cells.

[0087] <Creation of cancer stem cells transduced with drug-induced suicide genes> First, an IRES-iCaspase9-T2A-tdTomato construct was introduced into the LGR5 locus of a cultured organoid derived from colon cancer by the same method as in Experimental Example 1. Hereinafter, this organoid may be referred to as "LGR5-iCT organoid."

[0088] iCaspase9 is a drug-induced suicide gene. Although iCaspase9 has no activity as a monomer, it forms a dimer in the presence of AP20187 (dimerizer, APExBIO) and induces apoptosis. tdTomato is a type of red fluorescent protein (RFP).

[0089] Figure 4 is a diagram explaining the structure of the completed LGR5 locus and the behavior of the drug-induced suicide gene. In this system, iCaspase and tdTomato fluorescent protein are expressed by the promoter of the LGR5 gene. Therefore, the presence of LGR5-positive cells can be confirmed by observing the fluorescence of tdTomato. In the presence of AP20187, LGR5-positive cells undergo apoptosis and die. As a result, the fluorescence of tdTomato is no longer observed.

[0090] 《Confirmation of the behavior of LGR5-iCT organoids》 Using the same method as in Experimental Example 2, LGR5-iCT organoids were xenografted under the renal capsule of NOG mice (7-12 weeks old, male). After tumor formation, the mice were administered AP20187 daily for 5 days. In addition, control mice were administered only the solvent daily for 5 days. The tumor size of each mouse was then measured over time.

[0091] Figures 5(a) and (b) are fluorescent micrographs showing the results of tissue sections prepared by removing the tumors from each mouse on day 5 after the start of AP20187 administration, and detecting tdTomato fluorescence and the differentiation marker KRT20 by immunostaining.

[0092] In Fig. 5(a), "+Vehicle" indicates the results of control mice administered with the solvent alone. In Fig. 5(b), "+Dimerizer" indicates the results of mice administered with AP20187. In Fig. 5(a) and (b), the scale bars indicate 100 μm.

[0093] As a result, as shown in Figure 5(b), it was revealed that administration of AP20187 induced apoptosis and completely killed LGR5-positive cancer stem cells, whereas LGR5-negative cells remained intact.

[0094] 6 is a graph showing the results of measuring the tumor size over time in AP20187-administered mice (Dimerizer) and control mice (Vehicle). As a result, a decrease in tumor size due to administration of AP20187 was confirmed. However, it was revealed that the tumor size eventually increased again.

[0095] The inventors also investigated the expression of LGR5 gene in tumor tissues after administration of AP20187. As a result, it was revealed that the expression of LGR5 gene, which had disappeared once, was observed again. It was also revealed that the re-increase in tumor size corresponded to the re-expression of LGR5 gene.

[0096] These results demonstrate that LGR5-positive cancer stem cells are involved in tumor growth, and that LGR5-negative cancer cells have the ability to dedifferentiate into LGR5-positive cancer stem cells. In this experimental example, the cancer stem cell therapy model using AP20187 demonstrated that short-term treatment with a single drug that targets only cancer stem cells causes tumor recurrence. Furthermore, it was demonstrated that continuous cancer stem cell therapy can suppress tumor growth, although it does not cure the tumor.

[0097] [Experimental Example 5] (Investigation of a model for the combination of cancer stem cell-specific anticancer drugs and anticancer drugs) AP20187 can be considered a hypothetical cancer stem cell-specific anticancer drug in tumors created by xenografting of the LGR5-iCT organoids created in Experimental Example 4. Therefore, the effect of cancer treatment by combining AP20187 with existing anticancer drugs was examined.

[0098] Specifically, the LGR5-iCT organoids prepared in Experimental Example 4 were xenotransplanted under the renal capsule of NOG mice (7 to 12 weeks old, male) in the same manner as in Experimental Example 2. As the LGR5-iCT organoids, two clones, clone CCO7 and clone CCO25, were used.

[0099] After tumors were formed, the mice were administered an existing anticancer drug. The existing anticancer drug used was cetuximab (CTX), an anti-EGF receptor antibody. Cetuximab was administered twice, at the start of drug administration (day 0) and on day 7. Control mice were administered only the solvent instead of cetuximab.

[0100] The mice were then administered AP20187 daily for 5 days from the 8th to 12th day after the start of drug administration. For comparison, the mice were administered vehicle only daily for 5 days from the 8th to 12th day after the start of drug administration.

[0101] Subsequently, the tumor size of each mouse was measured over time. Figure 7(a) and (b) are graphs showing the results of measuring the tumor size over time. Figure 7(a) shows the results of the tumor derived from the LGR5-iCT organoid clone CCO7, and Figure 7(b) shows the results of the tumor derived from the LGR5-iCT organoid clone CCO25.

[0102] In Figures 7(a) and (b), "V" shows the results for control mice, "CTX" shows the results for mice administered cetuximab and vehicle, and "CTX+D" shows the results for mice administered cetuximab and AP20187.

[0103] The results showed that regardless of which organoid clone was used, the tumor size was significantly reduced by administering cetuximab and AP20187 in combination. In addition, tumor reduction of 95% or more was observed in 5 out of 6 mice transplanted with the CCO7 clone and administered cetuximab and AP20187 in combination, and complete disappearance of cancer tissue was observed in 5 out of 14 mice transplanted with the CCO25 clone.

[0104] These results indicate that the combination of cancer stem cell-specific anticancer drugs with existing anticancer drugs is effective for cancer treatment. It is believed that the drugs that have a combined effect differ depending on the cancer, and that the combined effect can be predicted from the expression level of LGR5 induced by the drug.

[0105] [Experimental Example 6] (Investigation of the effectiveness of anticancer drugs against cancer stem cells and cancer cells) A CMV-GFP construct was further introduced into the LGR5-iCT organoid prepared in Experimental Example 4. In the CMV-GFP construct, the GFP gene was linked downstream of the CMV promoter, which is a promoter for forced expression. Therefore, regardless of whether the LGR5 gene is expressed or not, the GFP gene is expressed in all cells, and the GFP protein emits fluorescence. Hereinafter, this organoid may be referred to as "LGR5-iCT / CMV-GFP organoid."

[0106] In the LGR5-iCT / CMV-GFP organoid, the survival activity of cancer cells can be observed by GFP fluorescence, and the survival activity of cancer stem cells can be observed by tdTomato fluorescence.

[0107] Using LGR5-iCT / CMV-GFP organoids, we performed high-throughput screening of existing anticancer drugs used in the treatment of colorectal cancer and examined their effects on cancer cells and cancer stem cells.

[0108] FIG. 8 shows micrographs of organoids showing representative results. In FIG. 8, "bright field" indicates a bright field observation image, "CMV-GFP" indicates a fluorescent micrograph in which GFP fluorescence was observed, "LGR5-tdTomato" indicates a fluorescent micrograph in which tdTomato fluorescence was observed, and "merge" indicates a result of combining a bright field observation image, a fluorescent micrograph of GFP, and a fluorescent micrograph of tdTomato. In addition, irinotecan, cetuximab, and oxaliplatin are existing anticancer drugs. Each anticancer drug was added to the organoid medium at the final concentration shown in FIG. 8. In addition, "DMSO" means dimethyl sulfoxide added to the medium instead of the anticancer drug as a control.

[0109] As a result, irinotecan was found to have an inhibitory effect on cancer cell proliferation, but no efficacy was observed against cancer stem cells. It was also revealed that the anti-EGFR antibody cetuximab significantly increased the survival activity of cancer stem cells. It was also revealed that oxaliplatin killed both cancer stem cells and cancer cells.

[0110] This result demonstrates that the method of this experimental example can be used to screen for anticancer agents and the like that are effective against cancer stem cells. [Industrial Applicability]

[0111] According to the present invention, a technique for performing locus-specific genetic manipulation on tissue stem cells can be provided.

Claims

1. A human tissue stem cell having a gene construct containing an Internal Ribosomal Entry Site (IRES)-target gene introduced into a locus of a stem cell marker gene, the stem cell marker gene is the Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) gene; A human tissue stem cell having self-renewal ability and differentiation ability, in which the gene construct is stably introduced into exon 18 of the LGR5 gene locus.

2. The human tissue stem cell according to claim 1 , wherein the target gene is a reporter gene.

3. The human tissue stem cell according to claim 1 or 2, wherein the target gene is a first reporter gene, and a second reporter gene driven by a promoter for forced expression is further introduced.

4. The human tissue stem cell according to claim 3 , wherein the second reporter gene is a gene encoding a cell lineage marker.

5. The human tissue stem cell according to any one of claims 1 to 4, which is a stem cell derived from cancer tissue.

6. A method for producing human tissue stem cells stably transfected with a gene of interest, comprising the steps of: A step of introducing a gene construct containing an IRES-target gene into the 18th exon of the LGR5 locus of human tissue stem cells in a cultured organoid by genome editing, the gene construct further comprising a gene cassette containing a drug resistance gene inserted downstream of another promoter sandwiched between LoxP downstream of the IRES-target gene; (2) selecting drug-resistant organoid clones; (3) removing the gene cassette in which a drug resistance gene has been integrated downstream of another promoter flanked by LoxP; As a result, human tissue stem cells into which a target gene has been stably introduced are obtained.

7. The method of claim 6, wherein the step (1) is performed by electroporation, and further comprises a step of culturing the cultured organoid at 30° C. for 2 days after the step (1).

8. The method according to claim 6 or 7, wherein the target gene comprises a gene encoding a lineage marking-inducing protein.

9. A method for testing whether a human tissue stem cell produced by the method of claim 8 is a stem cell, comprising a step of tracking and analyzing cells derived from a single cell by cell lineage analysis of the human tissue stem cell, and detecting self-renewal and differentiation of the human tissue stem cell, wherein detection of both self-renewal and differentiation of the human tissue stem cell indicates that the human tissue stem cell is a stem cell.

10. The method according to any one of claims 6 to 9, wherein the human tissue stem cells are stem cells derived from cancer tissue.

11. A method for screening an anticancer agent effective against human cancer stem cells, comprising: A step of culturing human cancer stem cells into which a reporter gene driven by the promoter of a human cancer stem cell marker gene has been introduced in the presence of a test substance, the human cancer stem cells being human cancer stem cells into which a gene construct containing an IRES-reporter gene has been introduced at the locus of the stem cell marker gene; measuring the expression level of the reporter gene, wherein a decrease in the expression level of the reporter gene indicates that the test substance is an anticancer agent effective against human cancer stem cells.

12. A method for testing the effectiveness of an anti-cancer drug against human cancer stem cells and human cancer cells, comprising: Culturing human cancer stem cells into which a first reporter gene driven by a promoter of a human cancer stem cell marker gene and a second reporter gene driven by a promoter for forced expression have been introduced in the presence of the anticancer drug; measuring the expression levels of the first and second reporter genes, wherein a decrease in the expression level of the first reporter gene indicates that the anticancer agent is an effective anticancer agent against human cancer stem cells, and a decrease in the expression level of the second reporter gene indicates that the anticancer agent is an effective anticancer agent against human cancer stem cells and human cancer cells; The method, wherein the first reporter gene is introduced into the gene locus of a cancer stem cell marker gene of the human cancer stem cell as a gene construct comprising an IRES-first reporter gene.

13. The human tissue stem cell according to claim 1 , wherein the target gene comprises a reporter gene and a drug-inducible suicide gene, and the human tissue stem cell is derived from a cancer tissue.

14. The human tissue stem cell according to claim 13, wherein the reporter gene is a first reporter gene, and a second reporter gene driven by a promoter for forced expression has been further introduced.

15. A method for verifying the effectiveness of an anticancer agent against human cancer stem cells and human cancer cells, comprising: Culturing the human tissue stem cell according to claim 14 in the presence of the anticancer agent; and detecting expression of the first and second reporter genes, wherein if only expression of the first reporter gene is detected, it indicates that the anticancer agent is an anticancer agent that is effective only against cancer cells, if only expression of the second reporter gene is detected, it indicates that the anticancer agent is an anticancer agent that is effective only against cancer stem cells, and if neither expression of the first reporter gene nor expression of the second reporter gene is detected, it indicates that the anticancer agent is an anticancer agent that is effective against human cancer stem cells and human cancer cells.

16. A method for screening an anticancer agent effective against human cancer stem cells and / or human cancer cells, comprising: Culturing the human tissue stem cell according to claim 14 in the presence of a test substance; a step of detecting expression of the first and second reporter genes, wherein if only the expression of the first reporter gene is detected, it indicates that the anticancer agent is an anticancer agent that is effective only against cancer cells, if only the expression of the second reporter gene is detected, it indicates that the anticancer agent is an anticancer agent that is effective only against cancer stem cells, and if neither the expression of the first reporter gene nor the expression of the second reporter gene is detected, it indicates that the anticancer agent is an anticancer agent that is effective against human cancer stem cells and human cancer cells.

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