JP2011087475A - Method for identifying undifferentiated cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for simply and accurately identifying undifferentiated cells containing stem cells such as multi-functional stem cells or cancer stem cells from cells or a cell group. <P>SOLUTION: A nucleic acid structure containing a reporter gene operably connected to Nanog promoter is transferred to cells or cell group of test control, and a reporter gene specific in an undifferentiated cell can thereby be expressed. Consequently, the objective undifferentiated cell can accurately be identified and detected. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for identifying undifferentiated cells including stem cells such as pluripotent stem cells and cancer stem cells. The present invention also relates to reagents and kits used for identifying undifferentiated cells including stem cells such as pluripotent stem cells and cancer stem cells. Furthermore, the present invention relates to an undifferentiated cell production kit used for producing undifferentiated cells containing stem cells such as induced pluripotent stem cells and cancer stem cells.

  Stem cells have the ability to differentiate into various tissues, and by utilizing their properties, various cells such as nerves, heart, pancreatic β cells and the like can be obtained in vitro. Furthermore, by using stem cells, it should be applied to the development of fundamental therapies for neurological and heart diseases that were difficult to treat in the past, and to drug discovery screening such as drug efficacy evaluation and safety testing in the field of drug discovery. Can do.

  In recent years, a technique has been reported in which somatic cells are initialized and induced into induced pluripotent stem (iPS) cells by introducing genes encoding OCT3 / 4, KLF4, c-MYC and SOX2 into somatic cells. Innovative technology is provided in the field of regenerative medicine (see Patent Document 1). This induced pluripotent stem cell is attracting attention as a useful cell resource because it can be prepared from tissues such as skin, and thus has low ethical barriers, and can be self-transplanted and avoid immune rejection.

  On the other hand, the present inventors have developed a technique for artificially producing induced cancer stem (iCS) cells from cancer cells based on a completely new idea that has not been heretofore (see Patent Document 2). Cancer stem cells show undifferentiated surface traits like normal stem cells, have self-renewal ability and differentiation ability, and have the property of producing all cancer cells in various differentiation stages constituting cancer tissue, Cancer stem cell preparation technology is extremely useful in accelerating the development of cancer drug discovery and diagnostic agents.

  However, in the production of induced pluripotent stem cells and induced cancer stem cells, one of the major problems that must be overcome for industrial application is low induction efficiency. In fact, induced pluripotent stem cells can be obtained only about 0.5 to 20% of induced cells, and induced cancer stem cells can be obtained only about 0.1 to 50% of treated cells. is there. Therefore, in the production of induced pluripotent stem cells and induced cancer stem cells, a technique for accurately distinguishing induced target stem cells is indispensable. In particular, in the production of induced pluripotent stem cells and induced cancer stem cells, in order to discriminate between induced target stem cells and cells that are merely immortalized, conventional methods include confirmation of the presence of cell surface markers, etc. We had to do it and it wasn't easy to do.

  Against the background of such conventional techniques, it has been desired to establish a technique for simply and accurately identifying undifferentiated cells including stem cells such as pluripotent stem cells and cancer stem cells from cells or cell populations.

International Publication No. 2007/069666 Pamphlet Japanese Patent Application No. 2009-241322

  An object of the present invention is to provide a technique for simply and accurately identifying undifferentiated cells such as pluripotent stem cells and cancer stem cells from a cell or a cell population. Furthermore, an object of the present invention is to provide an undifferentiated cell identification reagent, an undifferentiated cell identification kit, and an undifferentiated cell production kit using the technique.

  The inventors of the present invention conducted extensive studies to solve the above problems, and introduced a nucleic acid construct containing a reporter gene operably linked to a Nanog promoter into a test control cell or cell population. It was found that the reporter gene can be expressed specifically in undifferentiated cells, and as a result, the undifferentiated cells that are the target product can be accurately identified and detected. The present invention has been completed by further studies based on this finding.

That is, this invention provides the invention of the aspect hung up below.
Item 1. A method for identifying undifferentiated cells from a test cell or a test cell population, comprising:
(1) introducing a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter into a test cell or a test cell population, and
(2) A method for identifying an undifferentiated cell, comprising a step of identifying a cell expressing a reporter gene as an undifferentiated cell.
Item 2. Item 2. The identification method according to Item 1, wherein the reporter gene is a gene encoding GFP.
Item 3. Item 3. The identification method according to Item 1 or 2, wherein the nucleic acid construct used in the step (1) is a plasmid containing a reporter gene operably linked to a Nanog promoter.
Item 4. Any one of Items 1 to 3, which is a method for identifying an induced pluripotent stem cell obtained by reprogramming differentiated cells or an undifferentiated cancer cell obtained by reprogramming cancer cells containing differentiated cancer cells. The identification method described.
Item 5. An undifferentiated cell identification reagent comprising a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter.
Item 6. An undifferentiated cell identification kit comprising a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter.
Item 7. A kit for producing undifferentiated cells, comprising a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter.

  According to the present invention, undifferentiated cells including stem cells such as pluripotent stem cells and cancer stem cells can be easily and accurately identified and detected from a cell or a cell population. Therefore, according to the present invention, it is possible to accurately identify undifferentiated cells even for a cell population in which only a few undifferentiated cells are present. It becomes possible to obtain differentiated cells.

FIG. 1A shows the result of observing with a microscope the morphology of pluripotent stem cells derived from human fibroblasts (with the introduction of a GFP gene operably linked to the Nanog promoter); The result of observing an image with a fluorescence microscope; The result of observing with a microscope the morphology of human fibroblasts (with the introduction of a GFP gene operably linked to the Nanog promoter); The results observed with a fluorescence microscope are shown respectively. In addition, the lower right bar of A to D in FIG. 1 indicates 200 μm. FIG. 2A shows the result of microscopic observation of the morphology of undifferentiated cancer cells derived from human colon cancer cells (with the introduction of a GFP gene operably linked to the Nanog promoter); B shows the same results as A The result of observing an image with a fluorescence microscope; C shows the result of observing the morphology of a human colon cancer cell (with the introduction of a GFP gene operably linked to the Nanog promoter); D shows the same image as C The results of observation with a fluorescence microscope are shown respectively. In addition, the lower right bar of A to D in FIG. 2 indicates 200 μm. FIG. 2A shows the result of microscopic observation of the morphology of undifferentiated cancer cells derived from human cholangiocarcinoma cells (with the introduction of a GFP gene operably linked to the Nanog promoter); B shows the same results as A The result of observing an image with a fluorescence microscope; C shows the result of observing the morphology of a human cholangiocarcinoma cell (with introduction of a GFP gene operably linked to the Nanog promoter); D shows the same image as C The results of observation with a fluorescence microscope are shown respectively. In addition, the lower right bar of A to D in FIG. 2 indicates 200 μm.

1. Method for identifying undifferentiated cells The method for identifying undifferentiated cells of the present invention is a method for identifying undifferentiated cells from a test cell or a test cell population,
(1) introducing a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter into a test cell or a test cell population, and
(2) The method includes a step of identifying a cell expressing a reporter gene as an undifferentiated cell.

  Hereinafter, the identification method of the present invention will be specifically described.

  The identification method of the present invention is a method for identifying undifferentiated cells from a test cell or a test cell population. Here, the test cell is a cell for which determination of whether or not it is an undifferentiated cell is required, and the test cell population is required to determine whether or not an undifferentiated cell is included. Cell population. An undifferentiated cell is an undifferentiated state, a cell that can differentiate into various cells and has a self-proliferating ability. Specifically, it is a pluripotent stem cell (a variety of differentiated cells). Stem cells that can differentiate into cancer cells), cancer stem cells (stem cells that can differentiate into various cancer cells), and undifferentiated cancer cells related to cancer stem cells (undifferentiated cancer cells that arise from cancer stem cells, cancer stem cells) Undifferentiated cancer cells).

Further, the origin of the test cell or test cell population to be used in the identification method of the present invention is not particularly limited, regardless of whether it is a vertebrate or an invertebrate, and whether it is a mammal or a non-mammal. Absent. Suitable examples of such origin include, for example, those derived from mammals such as humans, mice, rats, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys; and birds derived from birds such as chickens and ostriches. The
If it is a thing.

  Some undifferentiated cells exist in a living body, and are obtained by initializing (reprogramming) differentiated cells to induce an undifferentiated state. The undifferentiated cells to be identified in the present invention may be those existing in the living body, or the differentiated cells (including normal cells and cancer cells) are initialized (reprogrammed) to be in an undifferentiated state. It may be induced by As a method for reprogramming differentiated cells, for example, a method of introducing each gene encoding OCT3 / 4, KLF4, c-MYC and SOX2 into differentiated cells is known.

  When the identification target is a pluripotent stem cell, as one embodiment of the identification method of the present invention, from a cell or a cell population obtained after performing a process of initializing differentiated cells and inducing pluripotent stem cells. A method for identifying the target pluripotent stem cell (induced pluripotent stem cell) is exemplified. Here, a technique for reprogramming differentiated cells to pluripotent stem cells is known, and for example, for differentiated cells (somatic cells), each gene encoding OCT3 / 4, KLF4, and SOX2, or Examples of the method of introducing each gene encoding OCT3 / 4, KLF4, c-MYC and SOX2 are exemplified, but the identification method of the present invention can be used to identify pluripotent stem cells prepared by any technique. Since detection is possible, the present invention is not limited to the pluripotent stem cells obtained by the method exemplified here.

  In addition, when the identification target is a cancer stem cell, as one embodiment of the identification method of the present invention, a cell obtained after performing a process of initializing a cancer cell (differentiated cancer stem cell) or Examples of the method include identifying a target cancer stem cell or undifferentiated cancer cell from a cell population. Here, the technique for reprogramming cancer cells to cancer stem cells is shown in Patent Document 2 described above. Specifically, for cancer cells, a KLF family gene (preferably KLF 4 gene), OCT family gene (preferably OCT3 / 4 gene), SOX family gene (preferably SOX2 gene), and at least one undifferentiation inducer selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS And / or a method for introducing an expression inhibitor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1. However, the identification method of the present invention is not limited to the cancer stem cells obtained by the method exemplified here because it can be identified and detected by any method.

  Furthermore, when the identification target is a cancer stem cell or an undifferentiated cancer cell, as one embodiment of the identification method of the present invention, a cancer stem cell or an undifferentiated cancer cell is obtained from a cancer cell or a cancer cell population obtained from a cancer tissue. The method of identifying is illustrated. Cancer stem cells or undifferentiated cancer cells are present only in a few percent or less in cancer cells constituting cancer tissue, but according to the present invention, cancer stem cells or undifferentiated cancer cells are present only in a small amount. Can be accurately identified and detected.

  In the identification method of the present invention, first, a nucleic acid construct containing a reporter gene operably linked to a Nanog promoter is introduced into a test cell or a test cell population (step (1)).

  The base sequence of the Nanog promoter is known. In the present invention, the origin and base sequence of the Nanog promoter are not particularly limited, but from the viewpoint of increasing the identification efficiency of undifferentiated cells, It is desirable to use the Nanog promoter that has the same origin as the differentiated cells. Specifically, when identifying human undifferentiated cells, it is preferable to use a human-derived Nanog promoter. Moreover, as long as the Nanog promoter used in the present invention functions as a Nanog promoter, a part of the base sequence of a conventionally known Nanog promoter may be modified. In particular, in the identification method of the present invention, when the Nanog promoter having the nucleotide sequence represented by SEQ ID NO: 1 is used, undifferentiated cells (particularly induced pluripotent stem cells, induced cancer stem cells, and induced undifferentiated cancer cells) can be obtained. It is preferable because the identification efficiency can be further increased.

  The reporter gene is not particularly limited as long as it encodes a reporter protein that can function as a detection marker. Specifically, green fluorescent protein (GFP), firefly luciferase, Renilla luciferase Examples thereof include genes encoding proteins such as β-galactosidase and alkaline phosphatase. The base sequences of these reporter genes are also known. Among these reporter genes, a gene encoding GFP is suitable in the present invention because it enables identification and detection of undifferentiated cells by a simple technique of the presence or absence of fluorescence. In addition, GFP-expressing undifferentiated cells can be recovered alive and can be used for biological or medical applications. From the standpoint of a gene encoding GFP as a reporter gene used in the present invention. Is preferable.

  A technique for operably linking a reporter gene to the Nanog promoter is also known in the art. Specifically, in order to obtain a reporter gene operably linked to the Nanog promoter, the 5 ′ end of the reporter gene may be directly linked to the 3 ′ end of the Nanog promoter, and the transcription control of the Nanog promoter is controlled. As long as it is possible, the 5 ′ end of the reporter gene may be linked to the 3 ′ end of the Nanog promoter via a linking region (base sequence serving as a linker).

  Further, the nucleic acid construct introduced into the test cell or the test cell population may be composed of a reporter gene operably linked to the Nanog promoter, but within the range that does not interfere with the effect of the present invention. In addition to the reporter gene operably linked to the promoter, it may contain a region consisting of another base sequence such as an enhancer.

  Introduction of the nucleic acid construct into a test cell or a test cell population can be performed by a known technique. Specifically, examples of a method for introducing the nucleic acid construct into a test cell or a test cell population include a method using a vector; a calcium phosphate method; a lipofection method; an electroporation method; a microinjection method and the like. Among these methods, a method using a vector is preferable from the viewpoint of improving the introduction efficiency and consequently improving the identification efficiency of undifferentiated cells. When the nucleic acid construct is introduced into a test cell or test cell population using a vector, a viral vector, plasmid vector, non-viral vector, artificial virus, or the like can be used as the vector. Such viral vectors and plasmid vectors are preferred.

  By incubating the test cell or test cell population into which the nucleic acid construct has been introduced for a period required for the expression of the reporter gene, for example, 6 to 72 hours, preferably 24 to 36 hours, only in undifferentiated cells, The reporter gene is expressed.

  In addition, undifferentiated cells are introduced by introducing genes that are initialized (reprogrammed) into undifferentiated cells (pluripotent stem cells, cancer stem cells, undifferentiated cancer cells) with respect to differentiated cells (normal somatic cells or cancer cells). When identifying an undifferentiated cell as a target product from a cell or a cell population obtained after performing a cell-inducing treatment, the nucleic acid construct is initialized (reprogrammed) into an undifferentiated cell. You may introduce | transduce into a differentiated cell with the gene of. Thus, by introducing a gene for initialization (reprogramming) together with the above-described nucleic acid construct into differentiated cells, induction into undifferentiated cells and identification / detection of undifferentiated cells can be performed in one step. This makes it possible to make the production and purification of undifferentiated cells easier.

  Next, after carrying out the above step (1), the cells expressing the reporter gene are identified as undifferentiated cells (step (2)). The method for confirming whether or not a reporter gene is expressed is also known in the art and is appropriately set according to the type of reporter gene. For example, when the reporter gene is a gene encoding GFP, the presence or absence of green fluorescence may be detected.

  Thus, cells in which the expression of the reporter gene is confirmed are identified as undifferentiated cells.

  The undifferentiated cells thus identified may be isolated as necessary, and the isolated undifferentiated cells can be used as a regenerative medicine, drug discovery screening tool or the like depending on the type. it can.

2. Reagents and kits for identifying undifferentiated cells As described above, identifying undifferentiated cells from a test cell or population of test cells by using a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter Is possible.

  Thus, the present invention provides an undifferentiated cell identification reagent containing a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter. Furthermore, the present invention also provides an undifferentiated cell identification kit containing a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter. In the present identification reagent and identification kit, the nucleic acid construct may be in a form suitable for introduction into a test cell or a test cell population, for example, a form incorporated in a vector. In addition, the identification kit includes, as necessary, a transfection reagent for introducing the nucleic acid construct into a cell, a manual with an experimental instruction book for carrying out the method for identifying the undifferentiated cell, and the like. May be included.

3. Undifferentiated cell production kit As described above, by using a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter, an undifferentiated cell derived from a somatic cell or a cancer cell (induced pluripotent stem cell, Induced cancer stem cells or induced undifferentiated cancer cells) can be detected.

  Therefore, the present invention further provides a kit for producing undifferentiated cells, which contains a nucleic acid construct containing a reporter gene operably linked to the Nanog promoter. According to this production kit, when undifferentiated cells (induced pluripotent stem cells, induced cancer stem cells or induced undifferentiated cancer cells) are induced from differentiated cells (normal somatic cells or cancer cells), the undifferentiated cells are accurately detected. It can be detected and isolated. In the production kit, the nucleic acid construct may be in a form suitable for introduction into a test cell or a test cell population, for example, a form incorporated in a vector. In addition, in this production kit, if necessary, a transfection reagent for introducing the nucleic acid construct into a cell; from a differentiated cell (normal somatic cell or cancer cell) to an undifferentiated cell (induced pluripotent stem cell, induction) Reagents for inducing cancer stem cells or induced undifferentiated cancer cells), including an instruction manual with experimental instructions for inducing undifferentiated cells from differentiated cells and identifying the induced undifferentiated cells It may be.

Hereinafter, the present invention will be described in detail based on examples and the like, but the present invention is not limited thereto.
Example 1: Identification of induced pluripotent stem (iPS) cells Human fibroblasts (HDF, Toyobo, Japan) were reprogrammed and introduced with a GFP gene operably linked to the Nanog promoter. Induced pluripotent stem (iPS) cells were prepared, identified and detected.

<Experiment method>
First, 1 × 10 ⁶ human fibroblasts are inoculated into 2 ml of 10% FBS-containing DMEM medium (containing 2 μg / ml puromycin) in a 30 mm dish, and 10 μl of Lipofectamine 2000 transfection reagent and a retroviral receptor are incorporated into the expression. 3-5 μg of lentiviral vector was added and incubated at room temperature for 20 minutes. Next, after removing the supernatant and washing the cells with 10% FBS-containing DMEM medium, 2 ml of 10% FBS-containing DMEM medium was added, and further 10 μl of Lipofectamine 2000 transfection reagent and the pMXs-based gene into which the gene shown below was incorporated. Each gene was transfected into human fibroblasts by adding 5 μg each of retroviral vectors (total 5 types) and incubating at 37 ° C. for 24 hours.
(Retroviral vector introduced)
PMXs-based retroviral vector incorporating a gene encoding OCT3 / 4 pMXs-based retroviral vector incorporating a gene encoding KLF4 pMXs-based retroviral vector incorporating a gene encoding c-MYC PMXs-based retroviral vector incorporating a gene encoding SOX2 • pMXs-based retroviral vector incorporating a nucleic acid construct in which the gene encoding GFP is directly linked to the 3 ′ end of the Nanog promoter (SEQ ID NO: 1). The cells after transfection were cultured for about 2 to 4 weeks while changing the medium every other day in a DMEM medium containing 10% FBS to induce pluripotent stem cells.

  After culturing, the presence or absence of GFP expression was observed with a fluorescence microscope along with observation of cell morphology.

In addition, as a control, only pMXs-based retroviral vectors incorporating a nucleic acid construct in which a gene encoding GFP is directly linked to the 3 ′ end of the Nanog promoter (SEQ ID NO: 1) were introduced into human fibroblasts. Except for the above, also in the case where the experiment was conducted in the same manner as described above, the presence or absence of GFP expression was observed with a fluorescence microscope together with the observation of the cell morphology.
<Experimental result>
The obtained results are shown in FIG. 1A shows the result of observing the morphology of pluripotent stem cells derived from human fibroblasts with a microscope; B shows the result of observing the same image as A with a fluorescence microscope; C shows only the GFP gene The result of having observed the form of the human fibroblast which introduce | transduced with a microscope; D shows the result of having observed the same image as C with the fluorescence microscope, respectively. As is clear from FIG. 1A, it was revealed from the cell morphology that human fibroblasts were induced into pluripotent stem cells from the cell morphology. The cells shown in FIG. 1A are confirmed to be pluripotent stem cells from the viewpoint of their cell growth characteristics and differentiation ability. From FIG. 1B, it was also confirmed that GFP was expressed in pluripotent stem cells derived from human fibroblasts. On the other hand, as can be seen from FIG. 1D, even when GFP was introduced into human fibroblasts under the control of the Nanog promoter, GFP expression was not observed.

  From the above results, it was confirmed that pluripotent stem cells can be specifically identified and detected by introducing a GFP gene operably linked to the Nanog promoter into pluripotent stem cells.

Example 2: Identification of cancer stem (iCS) cells Reprogramming of human colon cancer cells (DLD-1, donated by B. Vogelstein of John Hopkins University, USA) and human cholangiocarcinoma cells (HuCCT-1) At the same time, the GFP gene operably linked to the Nanog promoter was introduced to produce, identify and detect undifferentiated cancer cells.

<Experiment method>
First, 1 × 10 ⁶ cancer cells were inoculated into 2 ml of 10% FBS-containing DMEM medium (containing 2 μg / ml puromycin) in a 30 mm dish, and 10 μl of Lipofectamine 2000 transfection reagent and a retroviral receptor were incorporated into the expression. 3-5 μg of lentiviral vector was added and incubated at room temperature for 20 minutes. Next, after removing the supernatant and washing the cells with 10% FBS-containing DMEM medium, 2 ml of 10% FBS-containing DMEM medium was added, and further 10 μl of Lipofectamine 2000 transfection reagent and the pMXs-based gene into which the gene shown below was incorporated. Each gene was transfected into each cancer cell by adding 5 μg of each of retroviral vectors (total 5 types) and incubating at 37 ° C. for 24 hours.
(Retroviral vector introduced)
・ PMXs-based retroviral vector incorporating a gene encoding OCT3 / 4 ・ pMXs-based retroviral vector incorporating a gene encoding KLF4 ・ pMXs-based retroviral vector incorporating a gene encoding SOX2 ・ c- A pMXs-based retroviral vector incorporating a gene encoding MYC. A pMXs-based retroviral vector incorporating a nucleic acid construct in which a gene encoding GFP is directly linked to the 3 ′ end of the Nanog promoter (SEQ ID NO: 1). Each cancer cell after transfection is cultured in DMEM medium containing 10% FBS for 7 days while changing the medium every other day. After 7 days from transfection, mTeSRTM1 and Matrigel for BD human ES cells (StemCellTechnologies Inc) are used. The culture was performed while changing the medium every other day.

  After culturing, the presence or absence of GFP expression was observed with a fluorescence microscope along with observation of cell morphology.

As a control, only pMXs-based retroviral vectors incorporating a nucleic acid construct in which the gene encoding GFP is directly linked to the 3 ′ end of the Nanog promoter (SEQ ID NO: 1) were introduced into each of the above cancer cells. Except for the above, also in the case where the experiment was conducted in the same manner as described above, the presence or absence of GFP expression was observed with a fluorescence microscope together with the observation of the cell morphology.
<Experimental result>
The obtained results are shown in FIGS. 2A shows the result of observing the morphology of undifferentiated cancer cells derived from human colon cancer cells with a microscope; B shows the result of observing the same image as A with a fluorescence microscope; C shows the GFP gene. The result of having observed only the form of the human colon cancer cell which introduce | transduced only with the microscope; D shows the result of having observed the image same as C with the fluorescence microscope, respectively. 3A shows the result of observing the morphology of undifferentiated cancer cells derived from human cholangiocarcinoma cells with a microscope; B shows the result of observing the same image as A with a fluorescence microscope; The result of observing the morphology of human cholangiocarcinoma cells into which only the GFP gene was introduced with a microscope; D shows the result of observing the same image as C with a fluorescence microscope

  As shown in FIG. 2 and FIG. 3A, it was clarified from the cell morphology that human colon cancer cells and human cholangiocarcinoma cells were induced to undifferentiated cancer cells from the cell morphology. 2 and 3 are confirmed to be undifferentiated cancer cells from the viewpoint of their cell growth characteristics and differentiation ability. 2 and 3, it was also confirmed that GFP was expressed in undifferentiated cancer cells derived from human colon cancer cells and human cholangiocarcinoma cells. On the other hand, as shown in FIG. 2 and FIG. 3D, even when GFP was introduced into human colon cancer cells and human cholangiocarcinoma cell vesicles under the control of Nanog promoter, GFP expression was not observed. .

  From the above results, it was confirmed that undifferentiated cancer cells can be specifically identified and detected by introducing a GFP gene operably linked to the Nanog promoter into undifferentiated cancer cells.

Claims (7)

A method for identifying undifferentiated cells from a test cell or a test cell population, comprising:
(1) introducing a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter into a test cell or a test cell population, and
(2) A method for identifying an undifferentiated cell, comprising a step of identifying a cell expressing a reporter gene as an undifferentiated cell.   The identification method according to claim 1, wherein the reporter gene is a gene encoding GFP.   The identification method according to claim 1 or 2, wherein the nucleic acid construct used in the step (1) is a plasmid containing a reporter gene operably linked to a Nanog promoter.   4. The method according to claim 1, wherein the induced pluripotent stem cell is obtained by reprogramming a differentiated cell, or an undifferentiated cancer cell obtained by reprogramming a cancer cell containing a differentiated cancer cell. 5. The identification method described in 1.   A reagent for identifying undifferentiated cells, comprising a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter.   An undifferentiated cell identification kit comprising a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter.   A kit for producing undifferentiated cells comprising a nucleic acid construct comprising a reporter gene operably linked to a Nanog promoter.

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