Detection method and detection reagent for undifferentiated pluripotent stem cells

Abstract

The problem addressed by the present invention is to provide a method for detecting undifferentiated pluripotent stem cells and a reagent for detection by measuring the amount of RNA expressed by MIR302CHG gene to enable detection of a small amount of remaining undifferentiated pluripotent stem cells.

Description

Technical Field

[0001] The present invention relates to a method for detecting undifferentiated pluripotent stem cells and a reagent for the detection. Background Art

[0002] The use of renal progenitor cells (nephron progenitor cells) induced and differentiated from human pluripotent stem cells such as human iPS cells (iPSCs) and human ES cells is being studied to treat renal diseases such as renal failure. However, in clinical applications, cell preparations prepared from iPS cells have some tumorigenicity issues caused by residual iPS cells. Therefore, for cell preparations, it is necessary to confirm that there are no residual iPSC cells to ensure the safety of cell preparations. However, it is very difficult to detect and evaluate a small amount of residual iPSC cells in a large amount of cell preparations. Therefore, it is very important to detect by identifying substances that are not present in nephron progenitor cells but are present in large amounts in iPS cells.

[0003] As a method for detecting residual iPS cells, detection methods such as detection by iPS cell culture expansion method (non-patent documents 1 and 2) and detection by PCR of LIN28A (non-patent document 3, etc.) have been reported. However, under the culture conditions used in the iPS cell culture expansion method, nephron progenitor cells are also efficiently cultured and expanded, so it is impossible to culture and expand iPS cells alone, and LIN28A is also expressed to a certain extent in nephron progenitor cells, and pancreatic and liver organoids differentiated from iPS cells, so these methods are difficult to use for iPS cell detection. Therefore, a new iPS cell detection method that can be used for nephron progenitor cells, pancreatic progenitor cells, or liver progenitor cells is needed. Prior art literature Patent Literature

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2013-158325 Non-patent literature

[0005] [Non-patent document 1] Watanabe T. et al., Cryotherapy 2021; 23:2:176-183

Non-patent document 2

Non-patent document 3

[0006] An object of the present invention is to provide a method and a reagent for detecting undifferentiated pluripotent stem cells such as residual iPS cells present in a cell population such as nephron progenitor cells induced by differentiation of iPS cells. Solution to the problem

[0007] The present inventors conducted intensive studies to solve the above problems and found that a small amount of undifferentiated pluripotent stem cells remaining in a cell group induced to differentiate from pluripotent stem cells can be detected by measuring the amount of RNA expressed by the MIR302CHG gene, thereby completing the present invention.

[0008] The present invention provides the following contents. [1] A method for detecting undifferentiated pluripotent stem cells, the method comprising: (A) A step of measuring the amount of RNA expressed by MIR302CHG in a cell population that may contain undifferentiated pluripotent stem cells. [2] The method according to [1], wherein the pluripotent stem cells are artificial pluripotent stem cells (iPS cells). [3] The method according to [1] or [2], wherein the RNA expressed by MIR302CHG is splice variant ENST00000509938.1 and / or splice variant ENST00000505215.1. [4] The method according to [3], wherein the RNA expressed by MIR302CHG is a splice variant ENST00000509938.1. [5] The method according to any one of [1] to [4], wherein the RNA expressed by MIR302CHG comprises: (a) the nucleotide sequence described in SEQ ID NO: 2 or SEQ ID NO: 3, or (b) An RNA sequence having a nucleotide sequence that is 90% or more identical to the nucleotide sequence described in SEQ ID NO: 2 or SEQ ID NO: 3. [6] The method according to any one of [1] to [5], wherein the step (A) includes a step of confirming amplification by quantitative PCR or a step of confirming amplification by 2-step ddPCR (droplet digital PCR). [7] A method according to [6], wherein the step of confirming amplification by quantitative PCR is a step of using an oligonucleotide primer set having a sequence of one or more pairs selected from the combination of sequence numbers 4 and 5, sequence numbers 6 and 7, sequence numbers 8 and 9, sequence numbers 10 and 11, sequence numbers 12 and 13, sequence numbers 14 and 15, sequence numbers 16 and 17, sequence numbers 18 and 19, sequence numbers 20 and 21, sequence numbers 22 and 23, sequence numbers 24 and 25, sequence numbers 26 and 27, sequence numbers 28 and 29, sequence numbers 30 and 31, and sequence numbers 32 and 33. [8] The method according to [6], wherein the step of confirming the amplification property by two-step ddPCR is a step using an oligonucleotide probe comprising the nucleotide sequence recorded in sequence number 36 and an oligonucleotide primer set having a sequence that is a combination of sequence number 34 and sequence number 35. [9] According to the method described in any one of [1] to [8], the step (A) includes: taking the amount of RNA in a cell population composed only of pluripotent stem cells as 1, when the amount of RNA in the cell population is measured to be greater than 0.000001, it is determined that the undifferentiated pluripotent stem cells contained in the cell population have been detected.

[10] The method according to any one of [1] to [9], comprising, before the step (A), a step of concentrating a fraction containing the undifferentiated pluripotent stem cells by cell sorting using an undifferentiated pluripotent stem cell-specific antigen.

[11] The method according to

[10] , wherein the undifferentiated pluripotent stem cell-specific antigen is TRA-1-60.

[12] A method according to any one of [1] to

[11] , wherein the cell population comprises renal unit progenitor cells, pancreatic progenitor cells, hepatic progenitor cells, ureteric bud cells, mesenchymal progenitor cells, vascular endothelial cells, chondrocytes, bile duct progenitor cells, erythropoietin-producing cells, mesenchymal progenitor cells, cardiomyocytes, and / or lung cells induced by differentiation of pluripotent stem cells, or cells obtained at an intermediate stage of differentiation from pluripotent stem cells to any of these cells.

[13] The method according to any one of [1] to

[12] , wherein the cell population comprises nephron progenitor cells induced by differentiation from pluripotent stem cells or cells obtained at an intermediate stage of differentiation from pluripotent stem cells to nephron progenitor cells.

[14] A kit for detecting undifferentiated pluripotent stem cells, comprising (i) an oligonucleotide primer for measuring the amount of RNA expressed by MIR302CHG, or (ii) a combination of an oligonucleotide primer and an oligonucleotide probe.

[15] A kit according to

[14] , wherein the oligonucleotide primers of (i) are an oligonucleotide primer set having sequences of one or more combinations selected from the group consisting of SEQ ID NOs: 4 and 5, SEQ ID NOs: 6 and 7, SEQ ID NOs: 8 and 9, SEQ ID NOs: 10 and 11, SEQ ID NOs: 12 and 13, SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs: 24 and 25, SEQ ID NOs: 26 and 27, SEQ ID NOs: 28 and 29, SEQ ID NOs: 30 and 31, and SEQ ID NOs: 32 and 33, or the combination of the primers and the probe of (ii) is a combination of an oligonucleotide primer set having sequences of the combination of SEQ ID NOs: 34 and 35 and an oligonucleotide probe comprising the nucleotide sequence described in SEQ ID NO: 36. Effects of the Invention

[0009] According to the present invention, it is possible to detect undifferentiated pluripotent stem cells remaining in a cell population including nephron progenitor cells differentiated and induced by pluripotent stem cells. Thus, it is possible to evaluate the safety of products such as regenerative medicine (for cell therapy, transplantation organ reconstruction, etc.) including nephron progenitor cells derived from human iPS cells. In addition, it is expected that it can also detect residual undifferentiated pluripotent stem cells in other differentiated cell culture systems other than nephron progenitor cells. Therefore, it is also possible to be applied to other differentiated cells other than nephron progenitor cells derived from human iPS cells, for example, pancreatic progenitor cells, liver progenitor cells, ureteric bud cells, interstitial progenitor cells (for example, renal interstitial progenitor cells), vascular endothelial cells, chondrocytes, bile duct progenitor cells, erythropoietin producing cells, interstitial progenitor cells, cardiomyocytes, pneumocytes, etc. regenerative medicine products. BRIEF DESCRIPTION OF THE DRAWINGS

[0010]

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【 Figure 3Compared with cells on day 4 of the NPC differentiation protocol (D4C), human iPSCs showed extremely high expression of markers (log2FC<10 and FDR<10 -5 )

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[0011] Hereinafter, the present invention will be described. The method for detecting undifferentiated pluripotent stem cells of the present invention comprises: A step of measuring the amount of RNA expressed by MIR302CHG in a cell population that may contain undifferentiated pluripotent stem cells.

[0012] <Undifferentiated pluripotent stem cells> In the present invention, pluripotent stem cells refer to stem cells that have the multipotency to differentiate into a variety of cells existing in an organism and also have the ability to proliferate, including any cells that are induced to differentiate into somatic cells such as renal unit progenitor cells, pancreatic progenitor cells, liver progenitor cells, ureteric bud cells, mesenchymal progenitor cells (for example, renal mesenchymal progenitor cells), vascular endothelial cells, chondrocytes, bile duct progenitor cells, erythropoietin-producing cells, mesenchymal progenitor cells, cardiomyocytes, and lung cells. Pluripotent stem cells are not particularly limited, and include, for example, embryonic stem cells (ES cells), artificial pluripotent stem cells (iPS cells), embryonic stem cells derived from cloned embryos obtained by nuclear transplantation (ntES cells), sperm stem cells ("GS cells"), embryonic germ cells ("EG cells"), cultured fibroblasts, pluripotent cells derived from bone marrow stem cells (Muse cells), etc. Preferred pluripotent stem cells are iPS cells and ES cells. The source of pluripotent stem cells is preferably a mammalian source from primates and rodents, preferably a primate source, and more preferably a human source.

[0013] The preparation method of iPS cells is well known in the art and can be prepared by introducing reprogramming factors into any somatic cells. Here, reprogramming factors can be listed, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3Glis1, or genes or gene products such as miR302 / 367 clusters, and these reprogramming factors can be used alone or in combination. Examples of the combination of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, and WO2009 / 126252. 9 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells 26: 2467-2474, Huangfu D, et al. al. (2008), Nat. Biotechnol. 26: 1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al.(2008),Cell Stem Cell,3:475-479,Marson A,(2008),Cell StemCell,3,132-135,Feng B,et al.(2009),Nat.Cell Biol.11:197-203,RLJudson etal., (2009), Nat. Biotechnol., 27: 459-461, Lyssiotis CA, et al. (2009), Proc NatlAcad Sci USA. 106: 8912-8917, Kim JB. et al. (2009), Nature 461: 649-643, Ichida JK, et al. al.(2009),Cell Stem Cell 5:491-503, Heng JC,et al.(2010),Cell Stem Cell 6:167-74, Han J, et al. (2010), Nature 463:1096-1100, Mali P, et al. (2010), Stem Cells 28:713-720, Maekawa M, et al. (2011), Nature 474:225-9, Cell Stem Cell. 2011; 8:376-88. doi: 10.1016 / j.stem.2011.03.001. .

[0014] Somatic cells include, but are not limited to, any of fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature healthy or diseased somatic cells, and also include any of primary cultured cells, passaged cells, and established cell lines. Specifically, somatic cells include, for example, (1) neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells and other tissue stem cells (adult stem cells), (2) tissue progenitor cells, (3) blood cells (peripheral blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lung cells, kidney cells, fat cells and other differentiated cells.

[0015] Undifferentiated pluripotent stem cells are cells that have the characteristics of pluripotent stem cells such as pluripotency and proliferation, and refer to pluripotent stem cells that have not been induced to differentiate, or that have not been differentiated. For example, pluripotent stem cells that have not been differentiated can be cells that have not lost pluripotency or pluripotency and proliferation ability, although they have been induced to differentiate into any cell. That is, undifferentiated pluripotent stem cells can be pluripotent stem cells that remain in a cell population that has not been induced to differentiate, or that has been differentiated from pluripotent stem cells into any cell. Therefore, undifferentiated pluripotent stem cells can specifically be ES cells, iPS cells, nt-ES cells, GS cells, EG cells, Muse cells, etc. remaining in a cell population formed by differentiating and inducing pluripotent stem cells such as ES cells, iPS cells, nt-ES cells, GS cells, EG cells, Muse cells, etc. into any cells.

[0016] <Cell Group> In the present invention, the cell population that may contain undifferentiated pluripotent stem cells is not particularly limited as long as it has the potential to contain undifferentiated pluripotent stem cells. Preferably, it is a cell population obtained by inducing pluripotent stem cells to differentiate into a specific lineage, that is, a cell population induced by differentiation of pluripotent stem cells. It should be noted that it is known that undifferentiated pluripotent stem cells may remain in a cell group formed by differentiation induction of pluripotent stem cells. Therefore, the case where undifferentiated pluripotent stem cells remain in a cell group containing cells formed by differentiation induction of pluripotent stem cells can be cited. However, even if undifferentiated pluripotent stem cells do not actually remain in a cell group containing cells formed by differentiation induction of pluripotent stem cells, it is also regarded as a "cell group that may contain undifferentiated pluripotent stem cells" in the present invention.

[0017] The cell population that may contain undifferentiated pluripotent stem cells preferably includes nephron progenitor cells, pancreatic progenitor cells, hepatic progenitor cells, ureteric bud cells, mesenchymal progenitor cells (e.g., renal mesenchymal progenitor cells), vascular endothelial cells, chondrocytes, bile duct progenitor cells, erythropoietin-producing cells, mesenchymal progenitor cells, cardiomyocytes, and / or lung cells, more preferably includes nephron progenitor cells, ureteric bud cells, and / or mesenchymal progenitor cells (e.g., renal mesenchymal progenitor cells), and further preferably includes nephron progenitor cells.

[0018] In addition, the cell population that may contain undifferentiated pluripotent stem cells preferably includes nephron progenitor cells, pancreatic progenitor cells, hepatic progenitor cells, ureteric bud cells, mesenchymal progenitor cells (e.g., renal mesenchymal progenitor cells), vascular endothelial cells, chondrocytes, bile duct progenitor cells, erythropoietin-producing cells, mesenchymal progenitor cells, cardiomyocytes, and / or lung cells obtained by inducing differentiation of pluripotent stem cells, or cells produced in an intermediate stage of differentiation from pluripotent stem cells to any of these cells (e.g., mesoderm cells, endoderm cells, foregut cells, or cells after these cells). The present invention relates to a cell population comprising renal progenitor cells, ureteric bud cells, and / or mesenchymal progenitor cells (e.g., renal mesenchymal progenitor cells) obtained by inducing differentiation of pluripotent stem cells, or a cell population obtained in an intermediate stage of differentiation from pluripotent stem cells to renal progenitor cells, ureteric bud cells, and / or mesenchymal progenitor cells (e.g., renal mesenchymal progenitor cells), and further preferably a cell population comprising renal progenitor cells obtained by inducing differentiation of pluripotent stem cells, or a cell population obtained in an intermediate stage of differentiation from pluripotent stem cells to renal progenitor cells.

[0019] The method of inducing differentiation of pluripotent stem cells into renal unit progenitor cells, pancreatic progenitor cells, hepatic progenitor cells, ureteric bud cells, mesenchymal progenitor cells (e.g., renal mesenchymal progenitor cells), vascular endothelial cells, chondrocytes, bile duct progenitor cells, erythropoietin-producing cells, mesenchymal progenitor cells, cardiomyocytes, and / or pneumocytes can use any known method, for example, differentiation induction can be performed by adding appropriate differentiation inducing factors to the culture medium. As a method for inducing renal progenitor cells by differentiation from pluripotent stem cells, specifically, for example, the methods described in Tsujimoto, H. et al. (2020), Cell Rep. 31, 107476, WO2020 / 213734, Morizane, R. et al. (2015) Nat. Biotechnol. 33, 1193-1200, and Li, Z. et al. (2016) Cell Stem Cell 19, 516-529, 2016 can be cited. Specific examples of methods for inducing pancreatic progenitor cells by differentiation from pluripotent stem cells include the method described in Kimura, A. et al., (2020) Cell Chem. Biol. 27, 1561-1572.e7. Specific examples of methods for inducing differentiation of pluripotent stem cells into hepatic progenitor cells include the methods described in WO2016 / 104717, Kotaka, M. et al. (2017) Sci. Rep. 7, 1-13., and Ouchi, R. et al. (2019) Cell Metab. 1-11. doi: 10.1016 / j.cmet.2019.05.007. Specific examples of methods for inducing differentiation of ureteric bud cells from pluripotent stem cells include the method described in Mae, SI, et al. Cell Rep, 32 (2020), 10.1016 / j.celrep.2020.107963. Specific examples of methods for inducing differentiation of pluripotent stem cells into vascular endothelial cells and chondrocytes include the method described in Tsujimoto H, et al. Cell Rep. 2020 Apr 7; 31(1): 107476. doi: 10.1016 / j.celrep.2020.03.040. Specific examples of methods for inducing differentiation of pluripotent stem cells into mesenchymal progenitor cells include methods described in WO / 2023 / 017848 and Takasato M, et al. Nature. 2015; 526: 564-568. doi: 10.1038 / nature15695. Specific examples of methods for inducing bile duct progenitor cells by differentiation from pluripotent stem cells include the method described in Matsui S, et al. Stem Cell Res. 2019 Mar; 35: 101400. doi: 10.1016 / j.scr.2019.101400. Specific examples of methods for inducing differentiation of erythropoietin-producing cells from pluripotent stem cells include the method described in Katagiri N, et al. Sci Rep. 2021 Feb 16; 11(1): 3936. doi: 10.1038 / s41598-021-83431-6. Specific examples of methods for inducing differentiation of pluripotent stem cells into mesenchymal progenitor cells include the method described in Chen J, et al. PLoS One. 2013; 8: e75682. doi: 10.1371 / journal.pone.0075682. Specific examples of methods for inducing differentiation of pluripotent stem cells into cardiomyocytes include the method described in Banovich NE, et al. Genome Res. 2018; 28: 122-131. doi: 10.1101 / gr.224436.117. Specific examples of methods for inducing differentiation of pluripotent stem cells into lung cells include the methods described in Jacob A, et al. Cell Stem Cell. 2017; 21: 472-488.e10.doi: 10.1016 / j.stem.2017.08.014 or Kerschner LJ, et al. J Cell Mol Med. 2020; 24: 9853-9870.doi: 10.1111 / jcmm.15568.

[0020] <MIR302CHG> MIR302CHG is known as a gene that contains a gene cluster encoding miRNA miR-302 / 367 at its locus, but does not encode a protein. It is known that the RNA expressed by MIR302CHG includes three long noncoding RNA (lncRNA) variants: ENST00000510655.1 (variant 655), ENST00000509938.1 (variant 938), and ENST00000505215.1 (variant 215). That is, the RNA expressed by MIR302CHG includes the above-mentioned miRNA and its precursor, and the above-mentioned lncRNA.

[0021] The RNA expressed by MIR302CHG measured in the present invention is preferably lncRNA. In addition, it is preferably any one or more variants of the above three lncRNA variants expressed by MIR302CHG, more preferably variant 938 and / or variant 215, and even more preferably variant 938.

[0022] Specific examples of sequences obtained by removing the polyA sequence from the nucleotide sequences of variant 655, variant 938, and variant 215 are shown in SEQ ID NOs: 1, 2, and 3. Therefore, the RNA measured in the method of the present invention may be an RNA having a nucleotide sequence comprising a sequence described in SEQ ID NO: 1, 2, or 3, or a sequence substantially identical to a sequence described in SEQ ID NO: 1, 2, or 3. More preferably, the RNA measured in the method of the present invention is an RNA having a nucleotide sequence comprising a sequence described in SEQ ID NO: 2, or a sequence substantially identical to a sequence described in SEQ ID NO: 2. It should be noted that bases such as 5'-methylated cytosine may be modified in cells, and therefore RNA having a sequence containing such a modified base in a sequence described in SEQ ID NO: 1, 2, or 3, or a sequence substantially identical to a sequence described in SEQ ID NO: 1, 2, or 3 is also included in the measurement object. In the method of the present invention, complementary RNA or complementary DNA of RNA having a sequence recorded in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a sequence substantially identical to a sequence recorded in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 can also be measured.

[0023] In the present invention, "substantially identical sequences" refer to nucleotide sequences that have an identity of more than 80%, preferably more than 90%, more preferably more than 95%, further preferably more than 97%, and particularly preferably more than 99% with respect to the overall nucleotide sequence exemplified above, or a nucleotide sequence in which one or several nucleotides at one or several positions are substituted, deleted, modified or inserted. Furthermore, the above-mentioned "1 or several" specifically means preferably 1 to 20, more preferably 1 to 10, further preferably 1 to 5, and particularly preferably 1 to 3.

[0024] <Measurement of RNA Amount> In the present invention, "determination of the amount of RNA" means measurement of the amount of RNA, and even if the measured amount is below the detection limit, it is included in "determination of the amount of RNA". The method for measuring the amount of RNA is not particularly limited as long as it is a method that can measure the amount of RNA contained in the cell population, and a known method can be used. For example, a method for measuring using probe hybridization, a method for measuring using nucleic acid amplification method, a method for measuring using mass spectrometry, a method for measuring using sequencing, etc. can be cited. As a method for measuring using nucleic acid amplification method, for example, a method for confirming amplification by quantitative RT-PCR composed of a reverse transcription (RT) reaction and PCR, or a method for confirming amplification by a 2-step (RT) ddPCR composed of a reverse transcription (RT) reaction and a 2-step ddPCR, or a One-step ddPCR that directly confirms the amplification of RNA, etc. can be cited. The method for measuring the amount of RNA of the present invention preferably includes a method for confirming amplification by quantitative RT-PCR or a method for confirming amplification by 2-step (RT) ddPCR.

[0025] Specifically, for example, it is preferably carried out by extracting RNA from a cell population, synthesizing DNA from the extracted RNA by reverse transcription reaction, and confirming amplification by quantitative PCR, or confirming amplification by two-step ddPCR.

[0026] The method for extracting RNA from the cell population is not particularly limited as long as it is a method that can extract RNA expressed by MIR302CHG, and can be appropriately selected from known methods. Specifically, for example, the AGPC method can be cited. The extracted RNA can be total RNA or partial RNA containing RNA expressed by MIR302CHG.

[0027] In addition, it is preferred that the RNA extracted from the cell population be subjected to a treatment to remove DNA. The treatment to remove DNA can be appropriately selected from known treatment methods, and examples thereof include a treatment method that adds a DNA degrading enzyme (DNase).

[0028] Typically, the process of synthesizing DNA from extracted RNA by reverse transcription reaction is implemented using reverse transcriptase. The method for synthesizing DNA by reverse transcription reaction can be appropriately selected from known methods, preferably using primers with polyA sequence or random sequence as targets, and synthesizing by reverse transcriptase using extracted RNA as template, more preferably using primers with polyA sequence as targets, and synthesizing by reverse transcriptase using extracted RNA as template. Reverse transcription reaction can also be performed simultaneously with quantitative PCR or 2-step ddPCR described later.

[0029] The method for confirming amplification by quantitative PCR can be appropriately selected from known methods, for example, SYBR (trademark) Green method, TaqMan (trademark) method, etc. The process of continuously or simultaneously performing the above-mentioned step of synthesizing DNA from RNA by reverse transcription reaction and quantitative PCR is also called quantitative RT-PCR.

[0030] It is preferred to confirm at least the amplification of RNA expressed by MIR302CHG by quantitative PCR. In quantitative PCR, examples of oligonucleotide primers for amplifying RNA expressed by MIR302CHG, its DNA complementary sequence, or a portion of its RNA complementary sequence include a combination of sequence numbers 4 and 5, 6 and 7, 8 and 9, 10 and 11, 12 and 13, 14 and 15, 16 and 17, 18 and 19, 20 and 21, 22 and 23, 24 and 25, 26 and 27, 28 and 29, 30 and 31, and 32 and 33.

[0031] Therefore, to confirm the amplification property of RNA by quantitative PCR, it is preferred to use an oligonucleotide primer set having a sequence of one or more combinations selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, and SEQ ID NO: 32 and SEQ ID NO: 33.

[0032] The method of confirming amplification by two-step ddPCR is carried out by ddPCR using a combination of oligonucleotide primers and oligonucleotide probes. It is preferred to confirm the amplification of RNA expressed by MIR302CHG by 2-step ddPCR. In 2-step ddPCR, examples of oligonucleotide primers and oligonucleotide probes for amplifying RNA expressed by MIR302CHG, its DNA complementary sequence, or a portion of its RNA complementary sequence include an oligonucleotide primer set having a sequence of a combination of sequence number 34 and sequence number 35 and an oligonucleotide probe having a sequence described in sequence number 36.

[0033] Therefore, when confirming the amplification of RNA by two-step ddPCR, it is preferred to use, for example, a DNA containing the nucleotide sequence described in SEQ ID NO: 36 as an oligonucleotide probe and a DNA having a sequence that is a combination of SEQ ID NO: 34 and SEQ ID NO: 35 as an oligonucleotide primer set.

[0034] The amount of RNA can be determined based on amplification by quantitative PCR or 2-step ddPCR, etc.

[0035] The amount of RNA expressed by MIR302CHG can be evaluated by absolute amount (copy number), and when a certain amount or more, for example, 1 copy or more or 10 copies or more, is detected, it can be determined that undifferentiated pluripotent stem cells are present. In addition, the amount of RNA expressed by MIR302CHG can be normalized using the expression level of a housekeeping gene (e.g., the TATA-box binding protein (TBP) gene) and evaluated using its value (relative amount). When the value is above a certain amount, it can be determined that undifferentiated pluripotent stem cells are present.

[0036] On the other hand, the amount of RNA measured can be expressed as a relative value to the positive control. Specifically, for example, the measured value of the amount of RNA expressed by MIR302CHG in a cell population composed of pluripotent stem cells is set as 1 and expressed as a relative value. For example, the measured value of the amount of RNA expressed by MIR302CHG in a cell group (first cell group) composed of pluripotent stem cells with a certain number of cells can be taken as 1, and the measured value of the amount of RNA expressed by MIR302CHG in a cell group (second cell group) with the same number of cells as the first cell group that may contain undifferentiated pluripotent stem cells can be expressed as a relative value, and the presence or absence of undifferentiated pluripotent stem cells can be determined based on this value. For example, when the relative value is 0.000001 or more, 0.00001 or more, 0.0001 or more, or 0.001 or more, it can be determined that undifferentiated pluripotent stem cells are present.

[0037] The cell population used in the method of the present invention may be purified, concentrated or separated from any part before the step of measuring the amount of RNA. Specifically, for example, the cell population that may contain undifferentiated pluripotent stem cells may be purified, concentrated, cultured and expanded using an undifferentiated pluripotent stem cell-specific antigen as an index, or using an undifferentiated pluripotent stem cell-specific nucleotide as a target mRNA switch, or using culture conditions suitable for the proliferation and adhesion of undifferentiated pluripotent stem cells, etc., to concentrate and / or separate the part that may contain undifferentiated pluripotent stem cells. As undifferentiated pluripotent stem cell-specific antigens, proteins, glycopeptides, glycoproteins, glycolipids, sugar chains, etc. that are specific to undifferentiated pluripotent stem cells can be listed, preferably cell surface antigens that are specific to undifferentiated pluripotent stem cells, specifically, for example, TRA-1-60 can be listed. As an mRNA switch that targets undifferentiated pluripotent stem cell-specific nucleotides, for example, miR-302a response switch (Fujita Y et al. Science Advance 2022 Jan 7; 8 (1): eabj1793.) can be listed. As undifferentiated pluripotent stem cell-specific proliferation and adhesion conditions, for example, Essential-8 / LN521 culture expansion method (Tano et al., PLoS One. 2014 Oct 27; 9 (10): e110496) and the like can be cited. In addition, cell sorting can also be performed by using cell surface antigens of nephron progenitor cells, pancreatic progenitor cells, hepatic progenitor cells, ureteric bud cells, interstitial progenitor cells (e.g., renal interstitial progenitor cells), vascular endothelial cells, chondrocytes, bile duct progenitor cells, erythropoietin-producing cells, interstitial progenitor cells, cardiomyocytes, lung cells, etc. as indicators to remove these cells from the cell population, thereby concentrating undifferentiated pluripotent stem cells. The cell sorting method can select any known method, for example, a method using concentration based on magnetic beads can be cited. Concentration based on magnetic beads can be implemented, for example, by MACS.

[0038] <Safety Evaluation> By using the method of the present invention, undifferentiated pluripotent stem cells can be detected in products using a cell population that may contain undifferentiated pluripotent stem cells, such as products for regenerative medicine based on nephron progenitor cells derived from human iPS cells, thereby enabling safety evaluation.

[0039] The cell population determined by the method of the present invention as not containing undifferentiated pluripotent stem cells can be further evaluated for in vivo tumor formation.

[0040] <Kit for undifferentiated pluripotent stem cell detection> The present invention also relates to a kit for detecting undifferentiated pluripotent stem cells, comprising a reagent for measuring the amount of RNA expressed by MIR302CHG. Specifically, the kit of the present invention comprises (i) an oligonucleotide primer for measuring RNA expressed by MIR302CHG, or (ii) a combination of an oligonucleotide primer and an oligonucleotide probe for measuring RNA expressed by MIR302CHG.

[0041] The oligonucleotide primers of (i) are preferably, for example, oligonucleotide primer sets having sequences of one or more combinations selected from the group consisting of sequence numbers 4 and 5, sequence numbers 6 and 7, sequence numbers 8 and 9, sequence numbers 10 and 11, sequence numbers 12 and 13, sequence numbers 14 and 15, sequence numbers 16 and 17, sequence numbers 18 and 19, sequence numbers 20 and 21, sequence numbers 22 and 23, sequence numbers 24 and 25, sequence numbers 26 and 27, sequence numbers 28 and 29, sequence numbers 30 and 31, and sequence numbers 32 and 33. As described above, such a combined oligonucleotide primer set can be used to confirm the amplification of RNA by quantitative PCR. Therefore, the kit of the present invention may further contain a reagent for quantitative PCR.

[0042] The combination of the oligonucleotide primer and the oligonucleotide probe in (ii) above can be, for example, a combination of an oligonucleotide primer set having a sequence of a combination of sequence numbers 34 and 35 and an oligonucleotide probe having a sequence described in sequence number 36. As described above, such a combination can be used to confirm the amplification of RNA by 2-step ddPCR. Therefore, the kit of the present invention may further contain a reagent for 2-step ddPCR or 1-step ddPCR. [Example]

[0043] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following embodiments.

[0044] Pluripotent stem cells Human iPS cells (human iPSCs) were used as pluripotent stem cells. Human iPSCs were maintained by feeder-free culture on cell culture plates coated with iMatrix using Stem Fit AK02N medium (Ajinomoto) or Stem Fit AK03N medium (Ajinomoto). In addition, human iPSCs were passaged every 4 or 5 days using 0.5 mM EDTA / PBS (Thermo Fisher Scientific), and mycoplasma contamination was checked regularly.

[0045] Nephron progenitor cells Nephron progenitor cells (NPCs) were induced from human iPSCs by the method described in Tsujimoto, H. et al. (2020), Cell Rep. 31, 107476. In addition, cells on the fourth day after differentiation from human iPSCs by the above method were used for analysis as Day 4 cells (D4C).

[0046] Renal mesenchymal progenitor cells Renal interstitial progenitor cells (IPCs) are induced to differentiate from human iPSCs by the method described in WO / 2023 / 017848, specifically, by the following method. First, human iPSCs were treated with a 1:1 mixture of TrypLE Select Enzyme (Thermo Fisher Scientific) and 0.5 mM EDTA / PBS for 5 minutes and washed with PBS (-). Then, the cells were peeled off with a cell scraper and dissociated into single cells by gentle pipetting. One day before the start of differentiation, 0.5 mL of Stem Fit AK02N (Ajinomoto Healthy Supply) supplemented with 10 μM Y-27632 and 2.4 μL / mL iMatrix-511 (Matrixome) was added to the cells at 1.3×10 4 Cells / cm 2The cells were seeded at a density of 100 μg / mL in a 24-well plate (Corning). After 24 hours (day 0), 5 μM CHIR99021 (Wako), 10 nM RA (Sigma), 1 ng / mL BMP4 (Peprotech) and 100 ng / mL bFGF (Wako) were added to a serum-free differentiation medium (hereinafter referred to as basal medium) consisting of DMEM / F12Glutamax (Thermo Fisher Scientific), B27 supplement minus vitamin A (ThermoFisher Scientific) and 0.5×Penicillin / Streptomycin, and culture was started. After another 24 hours (day 1), 5 μM CHIR99021, 100 ng / mL bFGF and 1 ng / mL BMP7 (R&D Systems) were used for culture. On day 2, the medium was replaced with a basal medium containing 5 μM CHIR99021, 100 nM LDN193189, and 10 μM A83-01 (Wako). On day 3, the medium was replaced with a basal medium containing 5 μM CHIR99021, 30 ng / mL bFGF, 10 ng / mL activin A (R&D Systems), and 30 μM Y-27632 (Wako). On day 4, the cells were washed with PBS (-), treated with Accumax (Innovative Cell Technologies), dissociated into single cells by gentle pipetting, and plated at 2.0 × 10 in a 96-well low cell adhesion plate (Thermo Fisher Scientific). 4 The cells were seeded at a density of 100 cells / well (3D culture). The culture medium used a basal medium containing 5 μM CHIR99021, 30 ng / mL bFGF, 10 ng / mL activin A, and 30 μM Y-27632, and cultured for 48 hours. After that, the cells were treated with a combination of 1 μM CHIR99021, 10 μM SB431542, 100 nM RA, 10 ng / mL IL-1b, and 500 nM smooth (SMO) agonist (SAG) for 48 hours, and then treated with a combination of 1 μM CHIR99021, 10 μM SB431542, 100 nM RA, and 500 nM smooth (SMO) agonist (SAG) for 72 hours for culture.

[0047] Ureteric bud cells Ureteric bud cells (ND) were induced from human iPSCs using the method described in Mae, SI, et al. Cell Rep, 32 (2020), 10.1016 / j.celrep.2020.107963.

[0048] RNA-Seq analysis Total RNA was isolated using RNeasy Mini kit (QIAGEN) according to the manufacturer's recommended protocol. Then, a sequence library was made using TruSeq Stranded Total RNA (Illumina). The library was sequenced using NovaSeq SPReagent Kit v1.5 (200 cycles) (Illumina) with 101-8-8-101 cycles. Quantification of gene expression was performed using the analysis pipeline (2.3.4) used in the ENCODE Project (https: / / www.encodeproject.org / pipelines / ENCPL002LPE / ). The reference sequence used GRCh38 in ENSEMBL 104. The gene used GRCh38 of GENCODE38. The comparative analysis of human iPSCs and NPCs was performed using two sets of analysis (DESeq2) using the values ​​(STAR-RSEM) calculated by the aforementioned pipeline.

[0049] The following procedures were used to analyze RNA-Seq data that had been previously reported and published in the database. For NPC and human iPSC (strain 201B7), the Homo sapiens GRCh38 cDNA cDNA gene annotation (Ensembl) was used, and transcripts were quantified using Salmon. The output results from Salmon were processed using tximport of the R / Bioconductor package to obtain gene expression values. DEG analysis was implemented using iDEP (Version 0.92). Using iDEP, enrichment analysis of the top 1000 DEGs obtained by the k-means algorithm was performed using the biological process of Gene Ontology (GO). GREIN was used to obtain normalized counts per million (CPM) at the gene level or transcripts per million (TPM) at the transcript level from RNA-seq data of pancreatic progenitor cells and liver organoids.

[0050] Quantitative RT-PCR Quantitative RT-PCR was performed according to the following procedure Total RNA was isolated using RNeasy kit (QIAGEN) according to the manufacturer's recommended protocol. cDNA was synthesized by reverse transcription reaction from total RNA using ReverTraAce (Toyobo) and oligo dT according to standard protocols. Quantitative PCR was performed using QuantStudio 3 Real Time PCR System (Thermo Fisher Scientific) and TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio). Denaturation was performed at 95°C for 30 seconds, followed by 45 cycles of 95°C for 5 seconds and 60°C for 34 seconds. Data analysis of gene expression levels was quantified by the comparative Ct method, and the values ​​were normalized using TBP as a housekeeping gene. PCR was performed at least 3 times for each sample. The primers used for PCR are shown in Table 1.

Table 1

[0051] 2-step ddPCR A 2-step ddPCR was performed according to the following procedure. Total RNA was isolated using RNeasy kit (QIAGEN) according to the manufacturer's recommended protocol, and cDNA was synthesized from total RNA by reverse transcription using ReverTraAce (Toyobo) and oligo dT according to standard protocols. ddPCR was performed using ddPCR Supermix for Probes (No dUTP) (Bio-Rad), 900 nM forward and reverse primers, 250 nM probes labeled with FAM or HEX dye and ZEN-Iowa Black fluorescence quencher, and cDNA obtained from total RNA. First, droplets were generated in an 8-well box using a QX200 Droplet Generator (Bio-Rad) or an Automated Droplet Generator (Bio-Rad) according to the manufacturer's instructions. The water-in-oil emulsion was transferred to a 96-well plate and PCR was performed using a C1000 Touch thermal cycler (Bio-Rad). The reaction conditions were 40 cycles of enzyme activation at 95°C for 10 minutes, denaturation at 94°C for 30 seconds, and annealing / extension reaction at 57.1°C for 60 seconds. After PCR amplification, the product was denatured at 98°C for 10 minutes and cooled at 4°C until the fluorescence intensity was measured. The fluorescence intensity of each droplet obtained from the sample was measured using a QX200 DropletReader (Bio Rad). Positive droplets containing amplified products were distinguished from negative droplets and counted by applying a threshold of fluorescence amplitude using QuantaSoft software. The threshold was manually determined based on the distribution of droplets and was set to 4000 for TBP and 7000 for MIR302CHG. In QuantaSoft software, the concentration results are provided as copies of the target per 1 microliter (copies / μL). The ratio of the number of copies per TBP to the number of copies of the target gene was calculated.

[0052] Magnetic bead-based cell enrichment Magnetic bead-based cell concentration was performed using MACS (Miltenyi Biotec). Human iPSCs and NPCs were dissociated using Accumax (Innovative Cell Technologies, Inc.), and each cell suspension was centrifuged at 200 g for 7 minutes at 4°C. After centrifugation, the supernatant was discarded, and each cell pellet was resuspended in Rinsing solution (Miltenyi Biotec) supplemented with 0.5% BSA (Wako or Miltenyi Biotec) and 10 μM Y-27632 MACS. The mixture was prepared at a ratio of 1:10. 6 The ratio of human iPSC and NPC cell suspension is 10 6Add 1 μL of Anti-TRA-1-60 MicroBeads, human (Miltenyi Biotec) to each cell, incubate at 4°C for 15 minutes, and tap every 5 minutes. Then, filter the above suspension with a 100 μm cell strainer (Falcon) and load it onto an LS column equipped with a MidiMACS separator (Miltenyi Biotec). After that, wash the column twice with MACS Rinsing solution to which 0.5% BSA has been added. The loading and washing of the above labeled cells are performed by lightly pressing the syringe to make it fall at a rate of about 1 drop per 1 second. The positive fraction is recovered by lightly pressing the syringe at a rate of 2 to 3 drops per 1 second. After centrifugation at 200g for 5 minutes, the cells obtained from the positive and negative parts are each dissolved with RLT buffer (QIAGEN) to which β-mercaptoethanol has been added and stored at -80°C until analysis.

[0053] (Example 1) Detection of pluripotent stem cells To identify genes that are highly expressed in human iPSCs but not in NPCs, RNA-seq analysis including noncoding RNAs was performed, and 26 extremely highly expressed genes (log2FC<-10&FDR<10 -5 )( Figure 1 ). It was found that MIR302CHG, which is associated with lncRNA, showed a higher value of transcripts per million (TPM value) than LIN28A, which is currently used to detect residual iPS cells, and CUZD1, which has been confirmed to have differential high expression in human iPSCs in the analysis of stored RNA-seq data (data omitted) ( Figure 2 ).

[0054] In addition, MIR302CHG in iPSCs was found to be highly differentially expressed not only in NPCs after differentiation, but also in cells on day 4 (D4C) of the NPC differentiation protocol ( Figure 3 In addition, three splice variants of MIR302CHG, ENST00000510655.1 (variant 655), ENST00000509938.1 (variant 938), and ENST00000505215.1 (variant 215), were observed to be highly differentially expressed in the above iPSCs ( Figure 4 ).

[0055] Since the lncRNA expressed by MIR302CHG has a poly(A) 3' tail, reverse transcription (RT) reaction can be performed with oligo dT. Here, for MIR302CHG, CUZD1, and the pluripotency marker POU5F1, cDNAs derived from human iPSCs and NPCs or differentiation day 4 cells (D4C) were used, ranging from 100% to 10 -5 %10 -1 The analysis based on quantitative RT-PCR was performed. It should be noted that for MIR302CHG, the primers of sequence number 6 and 7 combination (corresponding to #2 in Table 1 above) were used. The results are shown in Figure 5 When the proportion of iPSCs was low, the expression pattern of MIR302CHG normalized by TBP was superior to that of CUZD1 and POU5F1 for the detection of iPSCs. This suggests that 10 -3 %(10 5 In D4C, 10 -1 (10 3 iPSCs (about 1 cell out of 10 cells) are mixed in.

[0056] In addition, human iPSCs and NPCs were analyzed by quantitative RT-PCR using the primers of each combination in Table 1 above, with lncRNA expressed by MIR302CHG as the target. The results are shown in Figure 6 For either combination of primers, iPSCs showed significantly higher comparative Ct (dCt) values ​​than NPCs.

[0057] (Example 2) Cell enrichment and quantitative PCR-based pluripotent stem cell detection Mix 10% of the total cell number in the NPC cell population -4 % iPSC, made into 10 -4 %iPSCs. 10 -4 % iPSCs were subjected to magnetic bead-based cell enrichment targeting the TRA-1-60 surface antigen, and the TRA-1-60 positive fraction (PF) and negative fraction (NF) were recovered.

[0058] For MIR302CHG, CUZD1, and POU5F1, in NPC, iPSC, 10 -4Amplification was confirmed by quantitative RT-PCR in each part of %iPSC, TRA-1-60PF and TRA-1-60NF. It should be noted that for MIR302CHG, the primer combination of sequence numbers 6 and 7 (corresponding to #2 in Table 1 above) was used. The results are shown in Figure 7 . It is believed that the combination of magnetic bead-based enrichment and quantitative RT-PCR for measuring the amount of RNA expressed by MIR302CHG can detect 10 -4 %, i.e. 10 6 On the other hand, even with similar methods, it was difficult to detect 10 iPSCs in either NPC or D4C or both in CUZD1 and POU5F1. 6 More than one iPSC was mixed in.

[0059] (Example 3) Detection of pluripotent stem cells based on two-step ddPCR For MIR302CHG, cDNA derived from human iPSCs and NPCs was used, ranging from 100% to 10 -5 %10 -1 The quantitative RT-PCR analysis was performed on the mixture of the difference. It should be noted that the primers of sequence numbers 34 and 35 and the probe of sequence number 36 were used, which used variant 938 expressed by MIR302CHG as the target. The results are shown in Figure 8 . Display by 10 -4 The incorporation of more than 10% iPSC resulted in a higher amount of RNA expressed by MIR302CHG than that of NPC, especially at 10 -2 Therefore, it is believed that the 2-step ddPCR for measuring the amount of RNA expressed by MIR302CHG can detect 10% or more of iPSCs mixed in NPCs. -4 %~10 -2 %, i.e. 10 6 ~10 4 One or more iPSCs are mixed in.

[0060] (Example 4) Expression of MIR302CHG in human iPSC-derived pancreatic progenitor cells and human iPSC-derived hepatocytes The expression of MIR302CHG in human iPSC-derived pancreatic progenitor cells and liver organoids was analyzed from publicly available RNA-seq datasets. Fig. 9For pancreatic progenitor cells derived from human iPSC or human iPSC reported in Kimura et al (2020), Cell Chem. Biol. 27, 1561-1572, Fig.10 TPM values ​​of each transcriptional variant of MIR302CHG obtained by analyzing the public RNA-seq datasets for human iPSCs or human iPSC-derived hepatocytes reported in Kotaka, M. et al., 2017, Sci. Rep. 7, 1-13. In both cases, MIR302CHG was highly expressed in iPSCs, similar to the comparison with NPCs, but the expression became almost unobservable after differentiation. Therefore, MIR302CHG can be used to detect undifferentiated cells even when differentiating into pancreatic and liver lineages.

[0061] (Example 5) Expression of MIR302CHG in neural and mesenchymal progenitor cells derived from human iPSCs The expression of MIR302CHG in neural and mesenchymal progenitor cells derived from human iPSCs was analyzed from publicly available RNA-seq datasets. Fig.11 TPM values ​​of each transcript variant of MIR302CHG obtained by analyzing the public RNA-seq dataset for neural progenitor cells and mesenchymal progenitor cells derived from human iPSCs or human iPSCs reported in Chen et al., 2013, PLoS One. 2013; 8: e75682. doi: 10.1371 / journal.pone.0075682. High expression of MIR302CHG was observed in iPSCs, but expression became almost unobservable after differentiation. Therefore, MIR302CHG can also be used to detect undifferentiated cells during differentiation into neural lineages.

[0062] (Example 6) Expression of MIR302CHG in human iPSC-derived cardiomyocytes The expression of MIR302CHG in human iPSC-derived cardiomyocytes was analyzed from an RNA-seq dataset available to the public. Fig.12 TPM values ​​of each transcript variant of MIR302CHG obtained by analyzing human iPSC-derived cardiomyocytes or human iPSC-derived cardiomyocytes using a public RNA-seq dataset reported in Banovich et al., 2018., Genome Res. 2018; 28: 122-131. doi: 10.1101 / gr.224436.117. High expression of MIR302CHG was observed in iPSCs, but expression became almost unobservable after differentiation. Therefore, MIR302CHG can also be used to detect undifferentiated cells during differentiation into myocardial lineage.

[0063] (Example 7) Expression of MIR302CHG in lung cells derived from human ES cells (ESCs) The expression of MIR302CHG in human ESC-derived lung progenitor cells or lung organoids was analyzed from publicly available RNA-seq datasets. Fig.13 TPM values ​​of each transcript variant of MIR302CHG obtained by analyzing human ESCs, lung progenitor cells derived from human ESCs or lung organoids reported in Jacob A et al., Cell Stem Cell. 2017; 21: 472-488.e10.doi: 10.1016 / j.stem.2017.08.014 using a public RNA-seq dataset. High expression of MIR302CHG was observed in iPSCs, but expression became almost unobservable after differentiation. Therefore, even when differentiating into lung cells, undifferentiated cells can be detected by MIR302CHG.

[0064] (Example 8) Expression of MIR302CHG in vascular endothelial cells derived from human iPSCs, IVF-ESCs, and nt-ESCs The expression of MIR302CHG in human iPSCs, IVF-ESCs, or nt-ESCs and vascular endothelial cells derived therefrom was analyzed from RNA-seq datasets released to the public. Fig.14 TPM values ​​of each transcription variant of MIR302CHG obtained by analyzing the public RNA-seq data set for human iPSCs, IVF-ESCs, nt-ESCs or vascular endothelial cells derived from human iPSCs, IVF-ESCs, nt-ESCs reported in Zhao et.al., Proc Natl Acad Sci USA. 2017; 114: E11111-E11120. High expression of MIR302CHG was observed in iPSCs, but expression became almost unobservable after differentiation. Therefore, even when differentiating into vascular endothelial cells, undifferentiated cells can be detected by MIR302CHG.

[0065] (Example 9) MIR302CHG expression in human ESCs, anterior primitive streak, embryonic endoderm, anterior foregut, posterior foregut, midgut / hindgut The expression of MIR302CHG in human ESCs and the anterior primitive streak, embryonic endoderm, anterior foregut, posterior foregut, and midgut / hindgut induced from them was analyzed. Fig.15 As shown, MIR302CHG was found to be expressed in ESC, anterior primitive streak and embryonic endoderm, but was expressed at a low level in the anterior foregut, posterior foregut, midgut / hindgut. Therefore, it was found that by measuring the amount of MIR302CHG, not only undifferentiated pluripotent stem cells can be detected, but also cells in the early stages of differentiation such as embryonic endoderm and anterior primitive streak can be detected, and they can be distinguished from cells after the foregut, midgut, and hindgut.

[0066] (Example 10) MIR302CHG expression in renal mesenchymal progenitor cells and ureteric bud cells According to the above-mentioned records of "renal interstitial progenitor cells" and "ureteric bud cells", renal interstitial progenitor cells (IPC) and ureteric bud cells (ND) were obtained from human iPSC, respectively. The IPC and ND were analyzed for MIR302CHG by quantitative RT-PCR in the human iPSC part (for IPC, N=4, for ND, N=5). It should be noted that the measured values ​​were normalized with TBP as a housekeeping gene (MIR302CHG / TBP). The results are shown in Fig.16 . The expression of MIR302CHG in IPCs was so low that it was undetectable. In NDs, the expression of MIR302CHG was extremely low, at 1 / 10,000 of that in iPSCs. That is, MIR302CHG, which was observed to be highly expressed in iPSCs, became almost unobservable due to differentiation into renal mesenchymal progenitor cells and differentiation into ureteric bud cells. Therefore, when differentiating into renal mesenchymal progenitor cells and ureteric bud cells, undifferentiated cells can also be detected by MIR302CHG. Industrial Applicability

[0067] According to the present invention, it is possible to detect undifferentiated pluripotent stem cells remaining in a cell group containing nephron progenitor cells differentiated and induced by pluripotent stem cells. Thus, it is possible to evaluate the safety of products such as regenerative medicine (cell therapy, transplantation organ reconstruction, etc.) including nephron progenitor cells derived from human iPS cells. In addition, it is expected that it can also detect residual undifferentiated pluripotent stem cells in other differentiated cell culture systems other than nephron progenitor cells. Therefore, it is also possible to be applied to other differentiated cells other than nephron progenitor cells derived from human iPS cells, for example, pancreatic progenitor cells, liver progenitor cells, ureteric bud cells, interstitial progenitor cells (for example, renal interstitial progenitor cells), vascular endothelial cells, chondrocytes, bile duct progenitor cells, erythropoietin producing cells, interstitial progenitor cells, cardiomyocytes, pneumocytes, etc. regenerative medicine products.

Claims

1. A method for detecting undifferentiated pluripotent stem cells, the method comprising (A) A step of measuring the amount of RNA expressed by MIR302CHG in a cell population that may contain undifferentiated pluripotent stem cells. 2 . The method according to claim 1 , wherein the pluripotent stem cells are artificial pluripotent stem cells (iPS cells).

3. The method of claim 1, wherein the RNA expressed by MIR302CHG is a splice variant ENST00000509938.1 and / or ENST00000505215.

1.

4. The method of claim 3, wherein the RNA expressed by MIR302CHG is a splice variant ENST00000509938.

1.

5. The method according to any one of claims 1 to 4, wherein the RNA expressed by MIR302CHG comprises: (a) the nucleotide sequence described in SEQ ID NO: 2 or SEQ ID NO: 3, or (b) An RNA sequence having a nucleotide sequence that is 90% or more identical to the nucleotide sequence described in SEQ ID NO: 2 or SEQ ID NO:

3. 6 . The method according to claim 1 , wherein the step (A) comprises a step of confirming amplification by quantitative PCR or a step of confirming amplification by two-step ddPCR (droplet digital PCR).

7. The method according to claim 6, wherein the step of confirming amplification by quantitative PCR is a step of using an oligonucleotide primer set having a sequence of one or more pairs selected from the combination of SEQ ID NOs: 4 and SEQ ID NO: 5, SEQ ID NOs: 6 and SEQ ID NO: 7, SEQ ID NOs: 8 and SEQ ID NO: 9, SEQ ID NOs: 10 and SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NOs: 14 and SEQ ID NO: 15, SEQ ID NOs: 16 and SEQ ID NO: 17, SEQ ID NOs: 18 and SEQ ID NO: 19, SEQ ID NOs: 20 and SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NOs: 24 and SEQ ID NO: 25, SEQ ID NOs: 26 and SEQ ID NO: 27, SEQ ID NOs: 28 and SEQ ID NO: 29, SEQ ID NOs: 30 and SEQ ID NO: 31, and SEQ ID NO: 32 and SEQ ID NO:

33.

8. The method according to claim 6, wherein the step of confirming amplification by two-step ddPCR is a step using an oligonucleotide probe comprising the nucleotide sequence recorded in sequence number 36 and an oligonucleotide primer set having a sequence that is a combination of sequence number 34 and sequence number 35.

9. The method according to any one of claims 1 to 4, wherein the step (A) comprises: When the amount of RNA in a cell population consisting only of pluripotent stem cells is determined to be 1 and the amount of RNA in the cell population is measured to be 0.000001 or more, it is determined that the undifferentiated pluripotent stem cells included in the cell population have been detected.

10. The method according to any one of claims 1 to 4, wherein: This method includes, before the step (A), a step of concentrating a fraction containing the undifferentiated pluripotent stem cells by cell sorting using an undifferentiated pluripotent stem cell-specific antigen. The method according to claim 10 , wherein the undifferentiated pluripotent stem cell antigen is TRA-1-60.

12. The method according to any one of claims 1 to 4, wherein the cell population comprises renal unit progenitor cells, pancreatic progenitor cells, hepatic progenitor cells, ureteric bud cells, mesenchymal progenitor cells, vascular endothelial cells, chondrocytes, bile duct progenitor cells, erythropoietin-producing cells, mesenchymal progenitor cells, cardiomyocytes, and / or pneumocytes induced by differentiation of pluripotent stem cells, or cells obtained in an intermediate stage of differentiation from pluripotent stem cells to any of these cells.

13. The method according to any one of claims 1 to 4, wherein the cell population comprises nephron progenitor cells induced by differentiation from pluripotent stem cells or cells obtained at an intermediate stage of differentiation from pluripotent stem cells to nephron progenitor cells.

14. A kit for detecting undifferentiated pluripotent stem cells, comprising (i) an oligonucleotide primer for measuring the amount of RNA expressed by MIR302CHG, or (ii) a combination of an oligonucleotide primer and an oligonucleotide probe.

15. The kit according to claim 14, wherein the oligonucleotide primers of (i) are an oligonucleotide primer set having sequences of one or more combinations selected from the group consisting of SEQ ID NOs: 4 and 5, SEQ ID NOs: 6 and 7, SEQ ID NOs: 8 and 9, SEQ ID NOs: 10 and 11, SEQ ID NOs: 12 and 13, SEQ ID NOs: 14 and 15, SEQ ID NOs: 16 and 17, SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs: 24 and 25, SEQ ID NOs: 26 and 27, SEQ ID NOs: 28 and 29, SEQ ID NOs: 30 and 31, and SEQ ID NOs: 32 and 33, or the combination of the primers and the probe of (ii) is a combination of an oligonucleotide primer set having sequences of a combination of SEQ ID NOs: 34 and 35 and an oligonucleotide probe comprising the nucleotide sequence described in SEQ ID NO: 36.

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