JPWO2012115270A1 - Method for selecting iPS cell clones and method for selecting genes used in the selection method - Google Patents

Abstract

An object of the present invention is to provide a method for selecting an iPS cell clone that is unlikely to undergo tumorigenesis even if differentiated, and a method for selecting a gene used in the selection method. In the above, by examining the expression level of at least one gene in any one of the first to tenth gene groups, iPS cell clones that are unlikely to undergo tumorigenesis even when differentiated are selected. In addition, in each of a plurality of first iPS cell clones that are not normally differentiated or that are susceptible to tumorigenesis even when differentiated and a plurality of second iPS cell clones that are less likely to be tumorigenic, each iPS cell in an undifferentiated state By examining the expression level of a gene belonging to a candidate gene group including one or more genes in a clone, a gene to be used in a method for selecting an iPS cell clone that hardly causes tumorigenesis even when differentiated is selected.

Description

  The present invention relates to a method for selecting an iPS cell clone and a method for selecting a gene used in the selection method.

  Since iPS cells (induced pluripotent stem cells) are pluripotent and can be prepared from differentiated cells, they are expected to be used for transplantation in regenerative medicine. However, even if iPS cells are differentiated and transplanted, there has been a problem that cells derived from transplanted iPS cells become tumors in vivo (Miura K. et al., Nat Biotechnol. 2009 vol.27). : p.743-5.).

  An object of the present invention is to provide a method for selecting an iPS cell clone that is unlikely to undergo tumorigenesis even when differentiated, and a method for selecting a gene used in the selection method.

The present invention comprises the following items.
(1) A method for selecting an iPS cell clone, the step of examining an expression level of at least one gene in any of the following first to twelfth gene groups in an undifferentiated iPS cell: Including methods.
(2) The selection method according to item 1, wherein the expression level is not normally differentiated, or the expression level of the gene in an iPS cell clone that is prone to oncogenesis even if differentiated, and oncogenesis Comparing the expression level of the gene in iPS cell clones that are unlikely to occur.
(3) The selection method according to item 1, wherein in the iPS cell clone, a step of examining expression levels of all genes belonging to any one of the first to twelfth gene groups, and the examined genes In the group, a step of examining the number of genes having abnormal expression levels, and a gene having abnormal expression levels in iPS cell clones that are not normally differentiated or that are susceptible to oncogenesis even if differentiated. And a step of comparing the number of genes with the number of genes having abnormal expression levels in iPS cell clones that are unlikely to undergo tumorigenesis.
(4) The selection method according to (3), wherein the expression level is determined to be abnormal by comparing with an expression level of a pluripotent stem cell that is unlikely to cause tumorigenesis.
(5) The selection method according to item 1, wherein in the iPS cell clone, a step of examining expression levels of all genes belonging to any one of the first to twelfth gene groups, and the examined genes A clustering analysis of the iPS cell clones using the expression level in the iPS cell clone and the expression level in the iPS cell clone that is not normally differentiated or is easily differentiated even when differentiated as an index, Investigating whether the iPS cell clone is normally differentiated from an iPS cell clone that is unlikely to undergo tumorigenesis, or is classified into an iPS cell clone that is likely to undergo tumorigenesis even if differentiated.
(6) The selection method according to (5), wherein clustering analysis of the iPS cell clone is performed using as an index a relative value with an expression level of a pluripotent stem cell that is unlikely to cause tumorigenesis. The method according to claim 7.
(7) A method for selecting genes whose expression levels are examined in order to select iPS cell clones, and a plurality of first iPS cell clones that do not differentiate normally or are susceptible to oncogenesis even after differentiation, and oncogenesis A step of examining expression levels of genes belonging to a group of candidate genes including one or more genes in undifferentiated iPS cells in a plurality of second iPS cell clones that are unlikely to cause morphogenesis.
(8) The method according to item 7, wherein the genes belonging to the candidate gene group are pluripotent stem cells that are higher in expression than differentiated cells and genes involved in DNA repair.
(9) The method according to item 7, wherein the gene belonging to the candidate gene group is a gene belonging to either the first gene group or the second gene group.
(10) The selection method according to item 7, wherein the first expression level obtained in the plurality of first iPS cell clones and the second expression obtained in the plurality of second iPS cell clones Comparing the level and examining whether the first expression level and the second expression level are different.
(11) The selection method according to item 7, wherein, in the expression level obtained in the plurality of first iPS cell clones, a first number of genes having an abnormal expression level, and a plurality of second Of the expression levels obtained in the iPS cell clone, a step of comparing the second number of genes having abnormal expression levels and a step of examining whether the first number and the second number are different from each other. Including methods.
(12) The selection method according to item 11, wherein the expression level is judged to be abnormal by comparing with an expression level of a pluripotent stem cell which is less likely to cause tumorigenesis.
(13) The selection method according to item 7, wherein the iPS cell clone is subjected to a clustering analysis using the expression level as an index, a plurality of first iPS cell clones and a plurality of second iPS. Examining whether each cell clone is classified into a different cluster.
(14) The selection method according to (13), wherein clustering analysis is performed on the iPS cell clone using the relative value to the expression level of pluripotent stem cells that are unlikely to be tumorigenic as an index. ,Method.

== Cross reference with related literature ==
This application claims priority based on Japanese Patent Application No. 2011-40979 filed on Feb. 25, 2011, and US Provisional Application US 61/466298 filed on Mar. 22, 2011. The application is hereby incorporated by reference.

In one embodiment of the present invention, the expression level of each iPS cell clone shown in Table 1 is 0.667 times or less or 1.5 times or more compared to the average expression level of human ES cells (KhES1-S3). It is the figure which graphed the number of the genes which show the abnormal expression level. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 2 in each clone in a figure. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 3 in each clone in a figure. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 4 in each clone in a figure. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 5 in each clone in a figure. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 6 in each clone in a figure. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 7 in each clone in a figure. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 8 in each clone in a figure. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 9 in each clone in a figure. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 10 in each clone in a figure. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 11 in each clone in a figure. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 12 in each clone in a figure. In one Example of the present invention, human iPS cells are induced to differentiate into tertiary neurospheres, transplanted into the brains of NOS / SCID mice, and analyzed in vivo for tumorigenicity. In one example of the present invention, human iPS cells (253G4) are induced to differentiate into tertiary neurospheres, transplanted into the brain of NOS / SCID mice, and the results of analyzing the proliferation of transplanted iPS cells in vivo are shown. It is. In one embodiment of the present invention, human iPS cells are induced to differentiate into tertiary neurospheres, injected into the testes of NOD / SCID mice, and after 3 months, teratomas are removed, (A) appearance, (B) long axis It is the figure which observed the length of the direction, (C) the expression of the reprogramming gene introduce | transduced at the time of iPS cell preparation. In one Example of this invention, it is a figure which shows the result of having performed the array CGH analysis with respect to the undifferentiated iPS cell and the tertiary neurosphere which differentiated each cell. (A) On the genomic DNA, the location where an abnormality is detected is indicated by an arrow. (B) The number of genes having abnormalities on the genomic DNA is shown in a graph. In one Example of this invention, it is a figure showing the tree obtained by performing clustering analysis using all the genes. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 4 in each clone in a figure. In one Example of this invention, it is the figure which showed the tree obtained by clustering analysis of the expression of the gene shown in Table 12 in each clone in a figure. In one Example of this invention, about the probe of a 12th gene group, it is a graph which shows the result of having measured the number of the probes which have a 1.5 times or more difference in signal intensity from the average value of the expression in ES cell. In one embodiment of the present invention, human iPS cells prepared using an episomal vector are induced to differentiate into tertiary neurospheres, injected into the testes of NOD / SCID mice, and teratomas are removed 3 months later. ) Observing the appearance and (B) measuring the length in the major axis direction. In one Example of this invention, it is the tissue slice image of the testis obtained by transplanting the neural stem cell derived from 409B2, and its enlarged view (A, B). In one Example of this invention, it is the tissue slice image of the testis obtained by transplanting the neural stem cell derived from 414C2, and its enlarged view (A).

Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples.
Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd .; Juan S. Bonifacino (Bethesda, Maryland); Mary Dasso (Bethesda, Maryland); JB Harford, J. L-Schwartz, KM Yamada (Ed.), Current Protocols in Cell Biology, John Wiley & Sons Ltd. The described method, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.
The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

(1) Method for producing iPS cells iPS cells can be produced by introducing reprogramming factors into differentiated cells and dedifferentiating them. (Takahashi K & Yamanaka S. Cell 2006 vol.126: p.663-676; Takahashi K, et al., Cell 2007 vol.131: p.861-872).

  The initialization method is not particularly limited, but it is preferably induced to have pluripotency and self-proliferation ability by introducing a nuclear reprogramming factor. For example, an initialization method described in International Publications WO2005 / 080598 and WO2007 / 069666 can be used.

  The nuclear reprogramming factor is not particularly limited, but preferably includes a combination of gene products of genes selected from each gene group of the Oct gene group, the Klf gene group, and the Sox gene group, and the efficiency of iPS cell establishment Then, it is more preferable to include a combination further including a gene product of a gene group. The genes belonging to the Oct gene group include Oct3 / 4, Oct1A, Oct6, the genes belonging to the Klf gene group include Klf1, Klf2, Klf4, Klf5, etc., and the genes belonging to the Sox gene group include Sox1 , Sox2, Sox3, Sox7, Sox15, Sox17, Sox18. Examples of genes belonging to the Myc gene group include c-Myc, N-Myc, and L-Myc.

  As a nuclear reprogramming factor, in addition to the above combination, a combination including a Nanog gene, a lin-28 gene, and / or a glis1 gene in addition to the genes of the Oct gene group and the Sox gene group can be mentioned. Further, in addition to any combination of these genes, p53 may be deleted. For example, the p53 gene may be knocked down by shp53. In addition, when introducing into a cell, in addition to the gene of the said combination, you may introduce | transduce other gene products, for example, an immortalization inducer etc. are mentioned.

  These genes are genes that are highly conserved in vertebrates, and in this specification, unless the name of an animal is indicated, the gene including a homolog is assumed. Moreover, even if it is a gene which has a variation | mutation including polymorphism, the gene which has a function equivalent to a wild-type gene product shall also be included.

  Furthermore, in addition to the combination of these genes, cytokines and compounds may be added to the medium as cofactors. Examples of cytokines in this case include SCF and bFGF, which can be substituted for c-Myc, and examples of compounds include valproic acid, which can be substituted for c-Myc gene and Klf4 gene.

(2) Method for selecting iPS cell clones The method for selecting iPS cell clones according to the present invention is based on a predetermined gene group such as the first to twelfth gene groups shown in Tables 1 to 12 in undifferentiated iPS cells. And a step of examining the expression level of at least one gene.
The method for examining the expression level of the gene is not particularly limited, and for example, total mRNA is extracted from undifferentiated iPS cells by a conventional method, and using that mRNA, Northern blotting is performed using the gene to be examined as a probe. By performing PCR after reverse transcription, the expression level of the gene can be known. When examining the expression of a plurality of genes simultaneously, it is preferable to use a DNA array. That is, the expression level of a gene can be known by hybridizing all labeled mRNA to a DNA array in which a probe for the gene to be examined is immobilized and analyzing the intensity of the obtained signal.
Until now, there has been no known method for predicting what kind of property the iPS cell shows when it is differentiated by examining the gene expression level of the undifferentiated iPS cell. However, in one embodiment of the present invention, whether the iPS cell is unlikely to undergo tumorigenesis by examining the expression level of a specific gene in an undifferentiated iPS cell and using the expression level as an index. It is possible to predict. Hereinafter, although the specific method is demonstrated about the prediction method, the method of this invention is not limited to the following. In this specification, when only saying that “iPS cell clones are less likely to cause oncogenesis”, it means that the clones are less likely to become tumors when differentiated in vitro or transplanted and differentiated in vivo. If it does not differentiate, it will not be included.

[1] Method for comparing the expression of a specific gene For at least one gene in a predetermined gene group, expression of the gene in an iPS cell clone that does not differentiate normally in advance or is susceptible to oncogenesis even after differentiation And the expression level of the gene in iPS cell clones that are less prone to oncogenesis are determined using one or more iPS cell clones, respectively, and the levels of these expressions (hereinafter referred to as subject expression) are: Comparison is made with the expression level of the gene in the iPS cell clone to be examined.

  Here, a method for determining whether a certain clone is likely to cause a tumor is not particularly limited. For example, neurospheres are differentiated from iPS cells (Okada et al., Stem Cells. 2008 vol.26: p.3086- 98; Miura K. et al., Nat Biotechnol. 2009 vol.27: p.743-5), transplanted into the striatum and testis of NOD / SCID mice, and examined the differentiation state of neurospheres 1-6 months later , If the tumor is formed, if it is formed, the size and number of the tumor, or the abnormal growth of the transplanted cells may be examined (Okada et al., Stem Cells. 2008 vol.26: p.3086-98; Miura K. et al., Nat Biotechnol. 2009 vol.27: p.743-5). The criteria for determining whether or not a tumor is likely to develop may be appropriately determined by those skilled in the art depending on the intended use of the iPS cells. For example, when used for transplantation for therapeutic purposes, the criteria may be strict. When studying, loose standards should be used. At this time, all types of tumors may be taken into account, but only specific tumors such as gliomas may be taken into account.

  The method of quantifying the control expression level is not particularly limited. From the list of expression levels in a plurality of clones, the average or mean ± standard deviation (or error) of the expression levels in a plurality of clones, the upper limit of the expression level in a plurality of clones As long as it is possible to determine which expression level the expression level examined in the clone you want to examine, such as a range to the lower limit, a value or range estimated or statistically calculated from the expression levels in multiple clones, It may be a numerical value. An appropriate comparison method can be selected according to the digitization method. For example, it is tested whether there is a significant difference between the examined expression level and the control expression level, or whether the examined expression level falls within the range of the control expression level. If it is determined that the examined expression level does not differentiate normally or matches the control expression level of a clone that is prone to oncogenesis even if differentiated, the clone does not differentiate normally or is differentiated and tumor If the clone is determined to be suitable for the control expression level in a clone that is unlikely to cause oncogenesis, or is determined not to be tumorigenic, and the clone is determined to be compatible with the control expression level in the clone that is unlikely to cause oncogenesis, the clone is an iPS cell that is unlikely to cause oncogenesis. Judge that there is.

[2] Method for comparing the number of genes with abnormal expression Alternatively, one or more iPS cell clones that are not normally differentiated or that are prone to oncogenesis even if differentiated and one or more iPS that are less likely to be oncogenic In the cell clone, the expression level of all genes in a predetermined gene group is determined, the number of genes having abnormal expression levels is calculated, and the number of genes having abnormal expression levels in each iPS cell clone is determined ( Hereinafter referred to as the number of controls). Then, the number of genes having abnormal expression levels in the iPS cell clones whose properties are to be examined is compared with the number of controls.

  Here, for one gene, clustering analysis may be performed using the results of investigation using a plurality of types of probes. However, the proportion of genes using a plurality of types of probes is preferably small, and less than 20% of the total. Preferably, it is less than 10%, most preferably 0%. Even if a plurality of types of probes are used for one gene, it is preferable that the number of types of probes is small, preferably three or less types of probes are used, and more preferably two types or less probes are used. Most preferred is when one type of probe is used. In addition, a probe in which a gene that can be detected is not specified may be used, but the number is preferably smaller, preferably less than 20%, more preferably less than 10%, more preferably 0%. Most preferably it is. In addition, it can be determined in the same manner as in [1] whether normal differentiation occurs or whether differentiation is likely to cause tumorigenesis.

  The method for determining whether or not there is an abnormality in the expression level is not particularly limited, but it is preferable to determine by comparing with a normal pluripotent stem cell clone. Here, the normal pluripotent stem cell clone may be a pluripotent stem cell clone that is unlikely to cause oncogenesis, and the pluripotent stem cell may be an ES cell or an iPS cell. And about each gene of a gene group, compared with the expression level in a normal pluripotent stem cell clone, below a predetermined ratio and the expression level above a predetermined ratio are made abnormal. For example, the rate of abnormality is 10% or less, 20% or less, 50% or less, 66.7% or less, 80% or less, or 120% or more, 125% or more, 133.3% or more, 150% or more, For example, 180% or more.

  Here too, the method of presenting the control number is not particularly limited, from a list of the numbers in multiple clones, the average or average ± standard deviation (or error) of the numbers in multiple clones, from the upper limit to the lower limit of the numbers in multiple clones Any display may be used as long as it can be determined whether the number of clones to be examined is suitable, such as a range or a value or range estimated or statistically calculated from the number of clones. . An appropriate comparison method can be selected depending on the display method. For example, it is tested whether there is a significant difference between the number examined and the number of controls, or whether the number examined is within the range of the number of controls. And if it is determined that the number examined does not differentiate normally or matches the control number of clones that are prone to tumorigenesis even if differentiated, the clone will not differentiate normally or will become tumoriable even if differentiated. If it is determined that the clone is not susceptible to oncogenesis, or is not likely to cause oncogenesis, and if it is determined to match the number of controls in a clone that is less likely to cause oncogenesis, the clone is determined to be less likely to cause oncogenesis. The

[3] Method of classifying by clustering analysis Alternatively, in one or more iPS cell clones that are not normally differentiated or that are susceptible to oncogenesis even after differentiation, Determine the expression level of all genes. Here, also for iPS cell clones whose properties are to be examined, all expression levels of the same gene group are determined, and clustering analysis of each iPS cell clone is performed.

  Here, for one gene, clustering analysis may be performed using the results of investigation using a plurality of types of probes. However, the proportion of genes using a plurality of types of probes is preferably small, and less than 20% of the total. Preferably, it is less than 10%, most preferably 0%. Even if a plurality of types of probes are used for one gene, it is preferable that the number of types of probes is small, preferably three or less types of probes are used, and more preferably two types or less probes are used. Most preferred is when one type of probe is used. In addition, a probe in which a gene that can be detected is not specified may be used, but the number is preferably smaller, preferably less than 20%, more preferably less than 10%, more preferably 0%. Most preferably it is. In addition, it can be judged in the same manner as in [1] whether it does not differentiate normally, is susceptible to oncogenesis even if differentiated, and whether there is an abnormality in the expression of genes in a predetermined gene group. .

  Here, when performing clustering analysis, it may be used as an index whether the examined expression level is abnormal. The method for determining whether the expression level is abnormal is the same as described in [2]. In addition, clusteringt is Cluster 3.0 (Freeware by the Department of DNA Information Analysis, Human Genome Center, University of Tokyo; MJL de Hoon, S. Imoto, J. Nolan, and S. Miyano: Open Source Clustering Software. Bioinformatics, 20 (9): 1453--1454 (2004) .; http://bonsai.hgc.jp/~mdehoon/software/cluster/) and Gene spring GX (Agilent).

  As a result of clustering analysis, when iPS cells whose properties are to be examined are classified as clones that do not differentiate normally or are susceptible to tumorigenesis even after differentiation, the clones do not differentiate normally or become tumorous even if differentiated. When it is determined that the clone is not a clone that is likely to cause tumorigenesis or is not likely to cause tumorigenesis, and is classified as a clone that is unlikely to cause tumorigenesis, the clone is determined to be an iPS cell that is unlikely to cause tumorigenesis.

(3) Gene selection method As described in (1) above, in an undifferentiated iPS cell clone, the expression level of at least one gene in a predetermined gene group such as the first to twelfth gene groups is determined. By investigating, it is possible to select iPS cell clones that are unlikely to be tumorigenic.

  In order to determine whether a certain clone is unlikely to undergo tumorigenesis, the expression level of which gene or which gene group is examined may vary depending on the origin of iPS cells. Can be easily determined. The determination method will be described below.

  First, a candidate gene for selecting whether or not it can be used as a gene for selecting a clone is selected. This selection method is not particularly limited, and may be selected at random, but is preferably selected from genes that are higher in pluripotent stem cells than differentiated cells and genes involved in DNA repair. More preferably, it is selected from the gene group. This makes it possible to select a gene for selecting a target clone with extremely high probability. The method for selecting candidate genes from these gene groups is not particularly limited, but as in the examples, pluripotent stem cell clones that are unlikely to undergo tumorigenesis and those that do not differentiate normally or are susceptible to tumorigenesis even when differentiated. It is preferable to select genes whose expression levels are significantly different from those of competent stem cell clones. A group of genes including one or more, preferably two or more genes selected in this way is defined as the Xth gene group.

  A gene having higher expression than a differentiated cell in a pluripotent stem cell can be easily examined by those skilled in the art using RT-PCR, microarray, or the like. The gene involved in DNA repair may be any gene involved in any one of the DNA repair processes, and may be a gene reported as such in a paper, patent publication, database, or the like. Good. These reports are retrieved from articles and patent publications that are accessible from databases such as PUBMED, each country (eg, Japan, Europe, USA) Patent Office, WIPO HP, GO (Gene Ontology), etc. be able to. In other words, genes involved in DNA repair are functions in PUBMED, papers, patent gazettes, and gene information included in the patent offices of various countries (for example, Japan, Europe, and the US), WIPO databases, and GO databases. Most preferably, at least one of the genes is a gene reported to be involved in DNA repair.

  Next, one or more, preferably a plurality of first iPS cell clones that do not differentiate normally or are susceptible to oncogenesis upon differentiation, and one or more, preferably a plurality of second, idiosyncratic cells that are less likely to cause oncogenesis. In iPS cells, the expression level of the gene belonging to the X gene group is examined in each undifferentiated iPS cell. Then, using the expression level as an index, the first iPS cell clone and the second iPS cell clone are compared. The comparison method is not particularly limited, but some specific examples will be described below.

[1] Method of comparing expression levels themselves For example, it is examined whether the expression level of the first clone is different from the expression level of the second clone.

  Here, the judgment that the expression level is different is made by examining the expression level of the gene of the X gene group in clones in which it is unknown whether tumorigenesis is unlikely to occur, and the clones are the first clone and the second clone. As long as it can be determined which one of the two is applicable, any criterion may be used. For example, the expression levels in the first and second clones may be expressed as a range of the lowest level and the highest level, respectively, and may be determined to be different when the ranges do not overlap. However, the present invention is not limited to these.

  Thus, if the expression levels in the first clone and the second clone are compared and if they differ, this X gene group is used as a gene group for selecting clones that are unlikely to cause oncogenesis. be able to.

[2] Method for comparing the number of genes whose expression level is different from that of pluripotent stem cells Or, among the first and second iPS cell clones in the X gene group, the expression level is different from that of normal pluripotent stem cell clones If the number of different genes is measured and these numbers are different, this X-th gene group can be used as a gene group for selecting iPS cell clones that are less likely to cause tumorigenesis. A normal pluripotent stem cell clone is a pluripotent stem cell clone that has been found to be less likely to cause tumorigenesis when transplanted in vitro or in vivo. If present, it may be an ES cell or an iPS cell.

  Here, the judgment that the number is different is that the expression level of the gene of the X gene group is similarly examined in the iPS cell clones that are unclear whether or not they are likely to be tumorigenic, and the expression levels are different from those of normal pluripotent stem cell clones. As long as it can be determined whether the number of genes is compatible with the first iPS cell clone or the second iPS cell clone, any criteria may be used. Here, the standard for determining that the expression level is different from that of a normal pluripotent stem cell clone can be performed by the same method as described in [1]. Then, for example, if the distribution ranges of the numbers do not overlap or the average values of the numbers are significantly different between the first iPS cell clone and the second iPS cell clone, this Xth The gene group can be used as a gene group for selecting iPS cell clones that are unlikely to cause tumorigenesis.

[3] Method of comparing the results of clustering analysis This method compares the expression levels of the genes belonging to the X gene group with the normal pluripotent cell clones in the first and second iPS cell clones, Using the comparison results, clustering analysis of iPS cells is performed, and if the first and second iPS cell clones are classified as different clusters, they are used as genes for selecting iPS cell clones that are unlikely to cause tumorigenesis. Can be used. As the clustering method, the same method as described in (1) [3] can be used.

(1) Evaluation of properties of used pluripotent stem cell clones In the examples, as human ES cell clones, three clones of KhES1 to KhES3 (Suemori et al. Biochem Biophys Res Commun. 2006 vol.345: p.926- 32.) As clones of human iPS cells, each clone of 201B6, 201B7, 253G1, 253G4 (Takahashi et al .. Cell. 2007 131: 861-72; Nakagawa et al. Nat Biotechnol. 2008 vol.26: p.101) -6.) Was used. The culture was carried out using a commonly used method (Suemori et al. Biochem Biophys Res Commun. 2006 vol.345: p.926-32.).

First, when these iPS cell clones were differentiated into neurospheres in vitro using a known method (International Publication WO2010 / 090007), only 201B6 did not form neurospheres. Neurospheres were normally formed from 253G1 and 253G4.
After three passages of the three strains in which neurospheres were formed, the third neurosphere was induced to differentiate into neural cells using a known method (International Publication WO2010 / 090007). Differentiated into nerve cells, glial cells hardly differentiated. As a control, KhES1 cells differentiated in vitro were used.
When the gene expression profile was examined by microarray, the neural stem cells derived from human iPS cells showed almost the same profile as the neural stem cells derived from human ES cells (see (6) Comparative Example described later). In the third neurosphere after differentiation of human iPS cells, undifferentiated markers such as TRA-1-60, TRA-1-81, NCAM and the like are hardly detected, and these human iPS cell clones have differentiation resistance. It was confirmed that almost no cells or undifferentiated cells remained.
In addition, three clones with neurospheres were analyzed electrophysiologically, but action potentials were recorded in the same manner as human ES cells, indicating that these three clones can differentiate into functional neurons. .

(2) Evaluation of tumorigenicity of the pluripotent stem cells used Here, these human iPS cell clones were induced to differentiate into tertiary neurospheres, and the cells were isolated and transplanted into the brain of NOS / SCID mice. The in vivo differentiation ability and tumorigenicity were analyzed.

First, each iPS by a known method (Okada et al., Stem Cells. 2008 vol. 26: p. 3086-98; Miura K. et al., Nat Biotechnol. 2009 vol. 27: p. 743-5). Human EB (Embryoid body) was prepared from the cells and then induced to differentiate into primary neurospheres. One iPS cell clone 201B6 did not differentiate normally. Here, a lentivirus expressing Venus (Okada et al., Stem Cells. 2008 vol.26: p.3086-98) was introduced to produce neural stem cells expressing Venus. In addition, the third neurosphere was generated by passage 2 times, separated into single cells, and 1-5 × 10 ⁵ cells were injected into the striatum of NOD / SCID mice (Ogawa et al.,). J Neurosci Res. 2009 vol.87: p.307-17; Miura K. et al., Nat Biotechnol. 2009 vol.27: p.743-5). Six months after cell injection, the brain was removed from the mouse, and the differentiation state of the transplanted cell was examined immunohistochemically using antibodies against each of the markers of nerve cells, astrocytes, oligodendrocytes. In vivo, it was differentiated into three nervous system cells. As for the types of differentiated neurons, TH positive catecholaminergic neurons, ChAT positive cholinergic neurons, and GAD67 positive GABAergic neurons were detected, and all clones had normal differentiation ability.

At the same time, when mouse brain sections were stained with HE, images of lower glioma were observed in mice brain transplanted with 253G1 and 253G4, and cells having nuclear atypia (indicated by wrinkles in the figure) were also observed (Fig. 13). In contrast, in the mouse brain transplanted with 201B7, normal brain tissue was observed as in the control KhES1 (FIG. 13).
In particular, when the area where venus was expressed on the brain tissue section was examined, signs of tumor formation were observed in 253G1 and 253G4, and a huge tumor derived from transplanted cells was observed particularly in the transplanted tissue of 253G4. (FIG. 14). In 201B7, the above was not observed in the transplanted cells, similar to the control KhES1.

Next, in order to analyze the remaining undifferentiated cells with higher sensitivity, the tertiary neurospheres are separated into single cells, and 1-5 × 10 ⁵ cells are injected into the testes of NOD / SCID mice. Three months later, the testis was removed, and the appearance (A), the length in the long axis direction (B), and the expression of the reprogramming gene introduced at the time of iPS cell production (C) were observed (FIG. 15). In 201B7, in all respects, it was similar to the control KhES1, but in 253G1, the cell proliferation was almost normal, but unlike the control KhES1, expression of the reprogramming gene (POU5F1 only) was observed. It was. In 253G4, cell proliferation was remarkable and expression of reprogramming genes (particularly POU5F1) was also significantly increased. Moreover, in the histochemical analysis, as in the brain tissue section, the mouse brain transplanted with 201B7 was a normal tissue like the control KhES1, but the mouse brain transplanted with 253G1 and 253G4 had lower glioma. Images were observed, especially 253G4, which was highly malignant.

  From the above, 201B7 is a clone that is less likely to cause tumor formation in vivo when transplanted into tissue, and 253G1 and 253G4 are considered to be clones that are likely to cause tumor formation in vivo. It shows that 201B7 takes the same pattern as ES cells and 253G1 and 253G4 take a different pattern from the expression pattern of the gene group.

(3) Classification of iPS cell clones using the first to tenth gene groups Here, a gene group that is higher in the pluripotent stem cells than a differentiated cell, a gene group involved in DNA repair, and a gene group common to them From these, various gene groups are extracted and their expression patterns in ES cells and iPS cells are examined in an undifferentiated state. Depending on the expression pattern, clones that are unlikely to cause tumorigenesis are not differentiated or differentiated normally. Shows that clones that are prone to oncogenesis can be distinguished.

[1] Extraction of gene group First, 22 types of human tissues (bone marrow, cerebellum, cerebrum) were expressed as genes that are expressed in pluripotent stem cells higher than differentiated cells using microarray Human U-133 Plus 2.0 Array (Affymetrix). Cortex, fetal brain, heart, kidney, liver, lung, pancreas, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, bronchi, uterus) and KhES1-KhES3 ES Compare expression levels of cell clones,
1. 1. q value of FDR (False Discovery Rate) is less than 0.05 2. All ES3 clones are p-flags All ES3 clones should be at least 5 times higher than the average human tissue expression level.
1340 probes (Table 1) satisfying the above three conditions (Table 1) (including 929 identified gene species; including 102 probes for which no corresponding gene is identified) are selected, and 929 identified gene species are selected as the first gene. Grouped. Here, the specified gene means a gene whose gene name is known and is counted as a single gene.

  From the first gene group, 1304 probes (Table 2) excluding probes in which the average expression level of ES cell clones of KhES1 to KhES3 and the expression level of 201B7 are more than twice (Table 906 identified gene types; A gene group consisting of 96 probes whose corresponding genes are not specified) was selected, and 906 specified gene types were designated as the second gene group.

  On the other hand, as a group of genes involved in DNA repair, human genes registered in DNA repair were extracted from a database of GO (Gene Ontology), and 313 genes related to DNA repair were obtained. Also in the literature (Wood et al., Science 2001 vol.291 p.1284; Wood et al., 2005 Mutation research vol.577 p.275; Negrini Nat Rev Mol Cell Biol. 2010 vol.113: p.220- In 163, 163 genes related to DNArepair were obtained, 332 were extracted without duplication, a third gene group was prepared, and 676 probes corresponding to them were selected (Table 3). Then, 83 probes (Table 4) (including 60 identified gene species) overlapping in the second gene group and the third gene group are selected, and the 60 identified gene species are selected as the fourth gene. Grouped.

  Furthermore, from the genes of the first gene group, the expression levels of all 201B6, 253G1, and 253G4 are 1.5 times or more higher than the average expression level of the ES3 clone, and the difference between the expression level of 201B7 and the average ES is 1 80 probes that are within 5 times (Table 5) (70 identified gene species; including 3 probes for which the corresponding gene is not identified) are selected, and 70 identified gene species are selected as the fifth From the genes of the first gene group, the expression level of all of 201B6, 253G1, and 253G4 is 1.5 times or more of the average expression level of ES3 clone, and further, all of ES3 clone and 201B7 72 probes (Table 6) with a difference from the average of the expression level of 1.5 or less (Table 6) (63 identified gene species; (Including 2 probes not selected) and 63 identified gene species as the sixth gene group; from the genes of the third gene group, all the expression levels of 201B6, 253G1, 253G4 are 59 probes (Table 7) (including 54 identified gene species) that are 1.5 times or more than the average of the expression level are selected, and 54 identified gene species are defined as a seventh gene group; From the genes of 7 genes group, 18 probes (Table 8) whose difference between the ES3 clone and 201B7 is 1.5 times less than the average expression level of ES3 clone (Table 8) 17) and the 17 identified gene types are designated as the 8th gene group; the expression level of any of 201B6, 253G1, and 253G4 from the genes of the 4th gene group is 13 probes (Table 9) (identified gene species 13) that are at least 1.5 times the average expression level of the gene and the difference between the expression level of 201B7 and the average of ES is within 1.5 times And the corresponding gene is the 9th gene group; from the gene of the 4th gene group, all the expression levels of 201B6, 253G1, 253G4 are 1.5 times the average of the expression level of the ES3 clone 5 probes (Table 10) (including 5 identified gene species) in which the difference between the expression level of 201B7 and the mean of ES is 1.5 times or less are selected, and the corresponding genes are selected. It was set as the 10th gene group.

<Analysis method using microarray>
Affymetrix U133plus2.0 was used as a microarray.
Normal human tissues (bone marrow, cerebellum, cerebral cortex, fetal brain, heart, kidney, liver, lung, pancreas, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, bronchi, uterus) RNA was purchased from Clontech, Ambion, Cell Applications, Stratagene. For other cells, total RNA was collected with Trizol (InVitrogen), and column purification was performed using RNAeasy mini kit (Quiagen). Further, the DNA was treated with Qiagen RNAase-free DNase to decompose contaminating DNA. The quality of RNA was confirmed using Bioanalyzer 2100 (Agilent).

[2] Method of comparing the number of genes having abnormal expression In this case, in the probe containing the genes of the first gene group, the expression level of each iPS cell was 0. 0 compared to the average expression level of the ES3 clone. The number of probes showing an abnormal expression level of 667 times or less or 1.5 times or more was examined, and the results are graphed in FIG.

  Significant in the number of probes that are abnormally expressed in iPS cells (201B7) that are unlikely to be tumorigenic and iPS cells that do not differentiate normally (201B6) or that are susceptible to tumorigenesis even after differentiation (253G1, 253G4) There was a big difference.

  Therefore, by examining the expression of the genes of the first gene group and examining the number of genes that are abnormally expressed in pluripotent stem cells that are unlikely to be tumorigenic, it can be determined whether or not tumorigenicity is likely to occur.

[3] Method of classifying by clustering analysis Here, the ratio of the expression level of each iPS cell to the average expression level of the ES3 clone is calculated for the probe containing the genes of the second to tenth gene groups, and Log2 (ratio ) (Table 2 to Table 10). Clustering analysis is performed on these data using Cluster 3.0, and the obtained results are displayed as a Java Treeview (Freeware by DNA Information Analysis Field, Human Genome Analysis Center, University of Tokyo; MJL de Hoon, S. Imoto, J. Nolan, and S. Miyano: Open Source Clustering Software. Bioinformatics, 20 (9): 1453--1454 (2004) .; http://bonsai.hgc.jp/~mdehoon/software/cluster/) Thus, a tree was produced (FIGS. 2 to 10).

  As shown in the trees of FIGS. 2 to 10, among the examined iPS cells, only 201B7 is classified into the same group as human ES cells, and 201B6, 253G1, and 253G4 are one group separately. It is classified.

  In this way, the gene group consisting of genes that are higher in the pluripotent stem cells than the differentiated cells and genes selected from genes involved in DNA repair are compared with the expression levels in the pluripotent stem cells that are unlikely to cause tumorigenesis. Thus, it is possible to classify into iPS cells that do not differentiate normally or are easily differentiated even if differentiated, and iPS cells that are unlikely to become tumorous. Therefore, by using this method, it is possible to clarify which group an iPS cell that is not likely to cause tumorigenesis belongs, and to determine whether the iPS cell is less likely to cause tumorigenesis.

[4] Method of comparing expression level itself For example, in this example, the expression level of suppressor of Ty 16 homolog (S. cerevisiae) described in Table 10 does not differentiate normally from ES3 clone (201B6), There is a significant difference from (253G1, 253G4), which tends to cause oncogenesis even after differentiation. The expression level of iPS cells (201B7) that are less likely to cause tumorigenesis is not different from that of the ES3 clone, and is significantly different from iPS cells that are likely to be tumorigenic.

  Thus, it can be determined whether iPS cells are unlikely to undergo tumorigenesis by comparing the expression levels themselves.

(4) Classification of iPS cell clones using the 11th to 12th gene groups Here, the expression shown in Table 2 and Table 3 for the probe containing each gene belonging to the 1st gene group and the 3rd gene group Based on the numerical value representing the level, the SAM test was performed between the ES3 clone and the 201B7 group and the 201B6, 253G1, 253G4 group, and probes having a significant difference at each gene group at p <0.1 or p <0.05. 27 (Table 11) (including 26 identified genes) and 19 (Table 12) (including 17 identified genes) were extracted, respectively, and 26 and 17 identified gene species were extracted from the 11th. The gene group and the 12th gene group were used. (3) Similar to [3], when clustering analysis was performed, as shown in the trees of FIG. 11 and FIG. 12, only 201B7 was classified into the same group as human ES cells among the examined iPS cells. 201B6, 253G1, and 253G4 were classified into one group separately.

  Thus, iPS cells that do not differentiate normally or are easily differentiated by iPS cells and iPS cells that are less likely to be tumoriZed by differentiation of expression levels having statistical significance are also obtained. Can be classified into cells.

(5) Mutation in genomic DNA The present invention is based on the discovery that abnormal expression of genes involved in DNA repair in undifferentiated iPS cells affects the tumorigenicity of differentiated iPS cells. is there. Hereinafter, as a mechanism thereof, abnormalities in genes involved in DNA repair in an undifferentiated state cause numerous wounds in DNA, which may cause iPS cells to not differentiate normally or iPS cells to differentiate. Sometimes indicates that it becomes tumorous.

  First, undifferentiated iPS cells (201B6, 253G1, 253G4, 201B7), tertiary neurospheres obtained by differentiating each cell, fibroblasts (HDF) from which iPS cells are produced, and human fetus as a control Genomic DNA was isolated from derived neural stem cells (hNSC) and patient glioma cell-derived cells (GDC) and subjected to array CGH analysis.

  Genomic DNA is extracted from each clone and from normal human male genome (Promega) as a control using the phenol chloroform method, DNeasy Blood & Tissue Kit or QIAamp DNA mini Kit (Qiagen), and using PicoGreen And quantified. As each sample, 500 ng to 1000 ng of double-stranded DNA was digested with restriction enzymes (AluI and RsaI) to fragment the DNA. Thereafter, using a Genomic DNA Enzymatic Labeling kit (Agilent), the DNA fragment was labeled with Cy3 or Cy5, one was used as a control, and the other was used as a sample. After purification, using Sureprint G3 human CGH microarray 4x180K (Agilent), following the attached protocol, hybridization, scanning using an Agilent scanner, and signal quantification using Feature extraction software were performed. The obtained data was detected for Aberration using Agilent Genomic Workbench. Among the detected Aberrations, the Aberration found in the control HDF (human skin fibroblasts) is highly likely to be a mere CNV (copy number variation), so it is excluded from the analysis and used as an aberrated gene between each clone. The number of genes was compared.

  As shown in FIG. 16, in ES cells and iPS cells (201B7) that are unlikely to undergo tumorigenesis, even if differentiated, the genome abnormality does not change as a whole, while it does not differentiate normally (201B6). However, in the case of being prone to oncogenesis (253G1, 253G4), genomic abnormalities increase with differentiation, and the level of abnormality is not as high as that of cultured cells derived from patient glioma (GDC21, GDC36, GDC40). It was much higher than ES cells and 201B7.

  Thus, in an undifferentiated iPS cell, if there is an abnormality in the expression of a gene involved in DNA repair, a DNA mutation that occurs during the differentiation process cannot be repaired, and therefore it cannot be differentiated normally, or even if it is differentiated. It is thought that the cells become tumorous.

(6) Comparative Example In this comparative example, all probes in which expression was detected in any clone were targeted, and the clustering analysis software called R was used (http://www.r-project.org/), Clustering analysis was performed by Ward's hierarchial clustering method and Eluclidian distance. The results are shown in FIG. 17, and iPS cells that are unlikely to undergo tumorigenesis unless they select an appropriate gene group as in the present invention, and iPS cells that do not differentiate normally or are susceptible to tumorigenesis even if differentiated. Could not be distinguished by cluster.

(7) Analysis of human iPS cells prepared using episomal vectors Human iPS cell lines 409B2 and 414C2 (Okita et al., Nat Methods 2011) prepared using episomal vectors were used as control cell lines for comparison. , KhES1, KhES2, KhES3, 201B7, 409B2, 414C2, 201B6, 253G4, 253G1 clones, and the fourth gene group and the twelfth gene group using the microarray in the same manner as in (3). Analysis and clustering analysis were performed. As a result, as shown in the trees of FIGS. 18 and 19, in both cases, 409B2 and 414C2 were classified into the same branches as ES cells and 201B7.
Further, for the 19 probes in the 12th gene group, the number of probes having a difference of 1.5 times or more in signal intensity was measured from the average value of expression in three ES cells. As shown in FIG. The number was within 6 for 409B2, 414C2, and 201B7, and 10 or more for 201B6, 253G4, and 253G1, and there was a significant difference between the two groups (p = 0.017 by t test).
Next, 409B2 and 414C2 were evaluated for tumorigenicity by the same method as in (2). As shown in FIG. 21, 409B2 and 414C2 were tested after testis transplantation, similar to ES cells and 201B7. No swelling was observed. In addition, paraffin sections of testis transplanted with 409B2 and 414C2 were prepared, and stained with HE, and histological images of teratomas derived from transplanted cells were observed. As shown in FIGS. 22 and 23, tumors were observed from 253G4 and 253G1. A well-differentiated nerve cell and glial cell were observed.
As described above, the gene group of the present invention is useful for classifying iPS cells into clones that do not differentiate normally, or that tend to undergo tumorigenesis even when differentiated, and clones that do not tend to undergo tumorigenesis.

[Table 1A] Ratio of the expression level of each ES cell clone and each iPS cell clone to the average expression level of human ES cell clone (KhES1-S3) in 1340 probes including the first gene group (Table 1B)

[Table 1B] First gene group

[Table 2A] Ratio of the expression level of each ES cell clone and each iPS cell clone to the average expression level of human ES cell clone (KhES1-S3) in 1304 probes including the second gene group (Table 2B)

[Table 2B] Second gene group

[Table 3A] Ratio of the expression level of each ES cell clone and each iPS cell clone to the average expression level of human ES cell clone (KhES1-S3) in 676 probes including the third gene group (Table 3B)

[Table 3B] Third gene group

[Table 4A] Ratio of the expression level of each ES cell clone and each iPS cell clone to the average expression level of human ES cell clones (KhES1-S3) in 83 probes including the fourth gene group (Table 4B)

[Table 4B] Fourth gene group

[Table 5A] Ratio of the expression level of each ES cell clone and each iPS cell clone to the average expression level of human ES cell clones (KhES1-S3) in 80 probes containing the fifth gene group

[Table 5B] Fifth gene group

[Table 6A] Ratio of the expression level of each ES cell clone and each iPS cell clone to the average expression level of human ES cell clones (KhES1-S3) in 72 probes including the sixth gene group (Table 6B)

[Table 6B] Sixth gene group

[Table 7A] Ratio of the expression level of each ES cell clone and each iPS cell clone to the average expression level of human ES cell clone (KhES1-S3) in 59 probes including the seventh gene group (Table 7B)

[Table 7B] Seventh gene group

[Table 8A] Ratio of the expression level of each ES cell clone and each iPS cell clone to the average expression level of human ES cell clone (KhES1-S3) in 18 probes including the eighth gene group (Table 8B)

[Table 8B] Eighth gene group

[Table 9A] Ratio of the expression level of each ES cell clone and each iPS cell clone to the average expression level of human ES cell clone (KhES1-S3) in 13 probes including the ninth gene group (Table 9B)

[Table 9B] Ninth gene group

[Table 10A] Ratio of the expression level of each ES cell clone and each iPS cell clone to the average expression level of human ES cell clones (KhES1-S3) in 5 probes including the 10th gene group (Table 10B)

[Table 10B] Tenth gene group

[Table 11A] Ratio of the expression level of each ES cell clone and each iPS cell clone to the average expression level of human ES cell clone (KhES1-S3) in 27 probes including the eleventh gene group (Table 11B)

[Table 11B] Eleventh gene group

[Table 12A] Ratio of the expression level of each ES cell clone and each iPS cell clone to the average expression level of human ES cell clone (KhES1-S3) in 19 probes including the 12th gene group (Table 12B)

[Table 12B] 12th gene group

  According to the present invention, it is possible to provide a method for selecting an iPS cell clone that is unlikely to undergo tumorigenesis even when differentiated, and a method for selecting a gene used in the selection method.

Claims (14)

A method for selecting iPS cell clones, comprising:
In an undifferentiated iPS cell clone, in the gene group of any of the first to twelfth gene groups, a step of examining the expression level of at least one gene;
Including methods. The selection method according to claim 1, comprising:
The expression level is compared with the expression level of the gene in iPS cell clones that are not normally differentiated or are susceptible to tumorigenesis even when differentiated, and the expression level of the gene in iPS cell clones that are less likely to be tumorigenic Process,
A method further comprising: The selection method according to claim 1, comprising:
In the iPS cell clone, examining the expression levels of all genes belonging to any one of the first to twelfth gene groups;
In the examined gene group, a step of examining the number of genes having abnormal expression levels;
The number of genes examined is the number of genes that are abnormal in expression level in iPS cell clones that are not normally differentiated or that are susceptible to oncogenesis even if differentiated, and the expression level in iPS cell clones that are less likely to be tumorigenic Comparing with the number of abnormal genes in
A method further comprising: The selection method according to claim 3, wherein
A method comprising determining whether or not the expression level is abnormal by comparing with an expression level of a pluripotent stem cell clone that is unlikely to cause oncogenesis. The selection method according to claim 1, comprising:
In the iPS cell clone, examining the expression levels of all genes belonging to any one of the first to twelfth gene groups;
About the examined gene group, the expression level in the iPS cell clone, the expression level in the iPS cell clone that is not normally differentiated or is easily differentiated even if differentiated, and the expression level in the iPS cell clone that is likely to be tumoriZed And a clustering analysis of iPS cell clones using
Investigating whether the iPS cell clone is classified into an iPS cell clone that is not normally differentiated or is susceptible to oncogenesis even if differentiated, and an iPS cell clone that is less likely to be tumorigenic;
Including methods. The selection method according to claim 5, comprising:
The method according to claim 7, wherein clustering analysis of the iPS cell clone is performed using a relative value with an expression level of a pluripotent stem cell clone that is unlikely to be tumorigenic as an index. A method for selecting a gene to examine an expression level in order to select an iPS cell clone,
In each of a plurality of first iPS cell clones that are not normally differentiated or that are likely to be tumoriated even if differentiated and a plurality of second iPS cell clones that are less likely to be tumorigenic, each iPS cell clone in an undifferentiated state Examining the expression level of a gene belonging to a candidate gene group comprising one or more genes,
Including methods.   The method according to claim 7, wherein the genes belonging to the candidate gene group are genes that are higher in the pluripotent stem cells than the differentiated cells and genes involved in DNA repair.   The method according to claim 7, wherein the gene belonging to the candidate gene group is a gene belonging to either the first gene group or the second gene group. The selection method according to claim 7, comprising:
Comparing a first expression level obtained in a plurality of first iPS cell clones with a second expression level obtained in a plurality of second iPS cell clones;
Examining whether the first expression level and the second expression level are different;
Including methods. The selection method according to claim 7, comprising:
Among the expression levels obtained in the plurality of first iPS cell clones, the expression level of the first number of genes having abnormal expression levels and the expression levels obtained in the plurality of second iPS cell clones Comparing the second number of abnormal genes;
Examining whether the first number and the second number are different;
Including methods. The selection method according to claim 11, comprising:
A method comprising determining whether or not the expression level is abnormal by comparing with an expression level of a pluripotent stem cell clone that is unlikely to cause oncogenesis. The selection method according to claim 7, comprising:
For the iPS cell clone, performing a clustering analysis using the expression level as an index,
Examining whether a plurality of first iPS cell clones and a plurality of second iPS cell clones are classified into different clusters;
Including methods. The selection method according to claim 13, comprising:
A method comprising performing clustering analysis on the iPS cell clone using the relative value of the expression level of a pluripotent stem cell clone that is unlikely to be tumorigenic as an index.