JP7370602B2 - Evaluation method for differentiation resistance of undifferentiated cells - Google Patents
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Description
本発明は、未分化細胞の分化抵抗性評価法に関する。 The present invention relates to a method for evaluating differentiation resistance of undifferentiated cells.
再生医療に用いる未分化多能性幹細胞の分化抵抗性、すなわち分化誘導した細胞集団における未分化細胞の残存/混入のしやすさを評価することは癌化リスクの観点から極めて重要である。 It is extremely important to evaluate the differentiation resistance of undifferentiated pluripotent stem cells used in regenerative medicine, that is, the ease with which undifferentiated cells remain/contaminate the differentiated cell population from the viewpoint of canceration risk.
未分化細胞の分化抵抗性(分化しにくさ)の関与についてはDNAメチル化および遺伝子発現の報告がある（非特許文献１、２）。 Regarding the involvement of differentiation resistance (difficulty in differentiation) of undifferentiated cells, there are reports of DNA methylation and gene expression (Non-patent Documents 1 and 2).
また、これまでに、分化細胞での未分化iPS細胞の残存/混入を検出、評価する手法は報告があるが（非特許文献３、４）、未分化iPS細胞の段階で未分化細胞の残存/混入リスクを評価する手法は存在しない。 In addition, there have been reports on methods to detect and evaluate the presence/contamination of undifferentiated iPS cells in differentiated cells (Non-patent Documents 3 and 4); /There is no method to assess the risk of contamination.
従来技術の中で、再培養法は混入する未分化iPS細胞からコロニーを形成させるため正確性が高いという利点が有る一方、検出までに1週間以上かかることから、定量PCRを用いた方法が簡便・迅速に実施可能な点で優れている。
また、分化細胞に未分化細胞が残存するか否かは、分化過程だけで無く、未分化iPS細胞の段階ですでに規定されていると考えられる。実際に、iPS細胞のクローンによって分化抵抗性があることが報告されており、分化抵抗性クローンに特徴的なメチル化や発現遺伝子も解析されている。
一方、本発明者らの研究から、分化能が高いiPS細胞クローンであっても継代を重ねることなどによって、分化能に変化が生じ、未分化iPS細胞が混入しやすくなる場合が有ることが明らかとなってきた。したがって、分化能が良好なクローンであっても、未分化iPS細胞混入リスクを日常的に評価することが重要である。
しかしながら、これまでに未分化iPS細胞の段階で分化細胞に未分化iPS細胞が混入する可能性を予測する手法は存在しなかった。
本発明は、未分化細胞の段階で、細胞が分化したときに未分化なままの細胞が残存/混入する可能性を予測する手法を提供することを目的とする。Among conventional techniques, the re-culture method has the advantage of being highly accurate as it forms colonies from contaminating undifferentiated iPS cells, but since it takes over a week for detection, a method using quantitative PCR is simpler.・It is excellent in that it can be implemented quickly.
Furthermore, whether or not undifferentiated cells remain among differentiated cells is considered to be determined not only during the differentiation process but also at the stage of undifferentiated iPS cells. In fact, it has been reported that some iPS cell clones are resistant to differentiation, and the methylation and expressed genes characteristic of differentiation-resistant clones have been analyzed.
On the other hand, research by the present inventors has shown that even if an iPS cell clone has a high differentiation potential, its differentiation potential may change due to repeated passages, making it easier for undifferentiated iPS cells to be mixed in. It has become clear. Therefore, it is important to routinely assess the risk of contamination with undifferentiated iPS cells, even for clones with good differentiation potential.
However, until now, there has been no method for predicting the possibility that undifferentiated iPS cells will be mixed into differentiated cells at the undifferentiated iPS cell stage.
An object of the present invention is to provide a method for predicting the possibility that undifferentiated cells will remain/contaminate cells when they are differentiated at the stage of undifferentiated cells.
本発明では未分化細胞を対象とし、分化誘導した際に分化細胞に未分化iPS細胞が残存/混入するリスクを評価するためのマーカー遺伝子の同定を行った。 In the present invention, targeting undifferentiated cells, marker genes were identified for evaluating the risk of undifferentiated iPS cells remaining/contaminating differentiated cells when differentiation is induced.
継代回数などが異なる同一iPS細胞クローンの分化細胞における未分化iPS細胞残存量を評価し、未分化iPS細胞が残存するiPS細胞と残存しないiPS細胞におけるDNAメチル化解析を実施し、DNAメチル化状態に差が有る遺伝子について、未分化残存と相関する遺伝子を特定した。
その結果、ZNF354C, C12orf56, ZNF578, MIR886は未分化iPS細胞が残存しないiPS細胞において発現が有り、DPP6は未分化iPS細胞が残存するiPS細胞において発現することを見出した。さらに、メチル化解析に用いなかった別のiPS細胞クローンにおいても、これらクローンの分化細胞における未分化iPS細胞残存の有無と、今回同定した遺伝子の発現に相関が有ることが明らかとなった。We evaluated the remaining amount of undifferentiated iPS cells in differentiated cells of the same iPS cell clone with different passage numbers, and conducted DNA methylation analysis in iPS cells with and without residual undifferentiated iPS cells. Among genes that differ in status, we identified genes that correlate with undifferentiated persistence.
As a result, it was found that ZNF354C, C12orf56, ZNF578, and MIR886 were expressed in iPS cells in which no undifferentiated iPS cells remained, and DPP6 was expressed in iPS cells in which undifferentiated iPS cells remained. Furthermore, even in other iPS cell clones that were not used for methylation analysis, it was revealed that there was a correlation between the presence or absence of residual undifferentiated iPS cells in the differentiated cells of these clones and the expression of the genes identified in this study.
これら遺伝子を用いることで、従来評価不可能であった未分化iPS細胞段階で、分化誘導した細胞への未分化iPS細胞の残存/混入の予測が可能となる。この手法は、iPS細胞だけでなく、ES細胞など他の未分化細胞についても応用可能である。 By using these genes, it becomes possible to predict the survival/contamination of undifferentiated iPS cells into differentiated cells at the undifferentiated iPS cell stage, which was previously impossible to assess. This method can be applied not only to iPS cells but also to other undifferentiated cells such as ES cells.
本発明の要旨は以下の通りである。
（１）下記の(i)及び／又は(ii)を測定することを含む、未分化細胞の分化抵抗性を評価する方法。
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態
（２）ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が高い場合に、未分化細胞の分化抵抗性が低いと評価し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が低い場合に、未分化細胞の分化抵抗性が高いと評価する（１）記載の方法。
（３）DPP6の発現レベル及び／又はプロモーター活性が高い場合に、未分化細胞の分化抵抗性が高いと評価し、DPP6の発現レベル及び／又はプロモーター活性が低い場合に、未分化細胞の分化抵抗性が低いと評価する（１）記載の方法。
（４）ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が低い(メチル化状態が高いと発現(=プロモーター活性)が低いため)場合に、未分化細胞の分化抵抗性が低いと評価し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が高い(メチル化状態が高いと発現が低いため)場合に、未分化細胞の分化抵抗性が高いと評価する（１）記載の方法。
（５）DPP6のプロモーターのメチル化状態が低い場合に、未分化細胞の分化抵抗性が高いと評価し、DPP6のプロモーターのメチル化状態が高い場合に、未分化細胞の分化抵抗性が低いと評価する（１）記載の方法。
（６）未分化細胞が、胚性腫瘍細胞（ＥＣ細胞）、胚性幹細胞（ＥＳ細胞）、人工多能性幹細胞（ｉＰＳ細胞）又は胚性生殖細胞（ＥＧ細胞)である（１）～（５）のいずれかに記載の方法。
（７）遺伝子の発現レベルをmRNAの量又はタンパク質の量として測定する（１）～（３）又は（６）のいずれかに記載の方法。
（８）遺伝子の発現レベルを測定する方法が、qPCR、デジタルPCR、免疫染色、in situ hybridization、RNAシークエンス、マイクロアレイ、NanoString、抗体アレイ、FlowCytometry、質量分析又はそれらの組み合わせである（７）記載の方法。
（９）遺伝子のプロモーターのメチル化状態を測定する方法が、メチル化 DNA を濃縮した後に、濃縮されたDNAを検出する方法、バイサルファイト処理による塩基置換した後の塩基配列を解読(シークエンス)する方法、バイサルファイト処理による塩基置換した後の塩基配列をハイブリダイゼーションにより検出する方法、メチル化特異的PCR(MSP)法、メチル化感受性の制限酵素による切断の有無で検出する方法、メチル化シトシンをグルコシル化し、グルコシル化シトシン感受性の酵素で検出する方法又はそれらの組み合わせである（１）、（４）～（６）のいずれかに記載の方法。
（１０）未分化細胞集団における、下記の(i)及び／又は(ii)を測定することによって、分化抵抗性の低いあるいは高い未分化細胞株を選別する方法。
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態
（１１）ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が高い場合に、分化抵抗性が低い未分化細胞株として選別し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が低い場合に、分化抵抗性が高い未分化細胞株として選別する（１０）記載の方法。
（１２）DPP6の発現レベル及び／又はプロモーター活性が高い場合に、分化抵抗性が高い未分化細胞株として選別し、DPP6の発現レベル及び／又はプロモーター活性が低い場合に、分化抵抗性が低い未分化細胞株として選別する（１０）記載の方法。
（１３）ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が低い場合に、分化抵抗性が低い未分化細胞株として選別し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が高い場合に、分化抵抗性が高い未分化細胞株として選別する（１０）記載の方法。
（１４）DPP6のプロモーターのメチル化状態が低い場合に、分化抵抗性が高い未分化細胞株として選別し、DPP6のプロモーターのメチル化状態が高い場合に、分化抵抗性が低い未分化細胞株として選別する（１０）記載の方法。
（１５）未分化細胞株が、胚性腫瘍細胞（ＥＣ細胞）株、胚性幹細胞（ＥＳ細胞）株、人工多能性幹細胞（ｉＰＳ細胞）株又は胚性生殖細胞（ＥＧ細胞)株である（１０）～（１４）のいずれかに記載の方法。
（１６）未分化細胞集団中に存在する、分化抵抗性の低いあるいは高い未分化細胞を検出するために、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子をマーカーとして使用する方法。
（１７）下記の(i)及び／又は(ii)を測定可能な試薬を含む、未分化細胞の分化抵抗性を評価するためのキット。
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態
（１８）遺伝子の発現レベルを測定可能な試薬が、プライマー、プローブ又は抗体である（１７）記載のキット。
（１９）遺伝子のプロモーター活性を測定可能な試薬が、プロモーター下流にレポータータンパク質を連結した遺伝子配列又はこの遺伝子配列を組み込んだベクターである（１７）記載のキット。
（２０）遺伝子のプロモーターのメチル化状態を測定可能な試薬が、バイサルファイト（亜硫酸水素塩）、メチル化解析用マイクロアレイ試薬、Sanger法によるシークエンス試薬、次世代シークエンサー用シークエンス試薬、5-mC抗体、5-hmC抗体、メチルアデノシン抗体、5’-methyl-2’-deoxycytidine抗体、HRP標識DNA抗体、5-hmC グルコシルトランスフェラーゼ、グルコシル-5hmC感受性制限酵素エンドヌクレアーゼ、MBD1 (Methyl-CpG Binding Domain Protein1)、MBD2 (Methyl-CpG Binding Domain Protein2)、特異的PCRプライマー、特異的プローブ又はDNA精製キットである（１７）記載のキット。The gist of the invention is as follows.
(1) A method for evaluating differentiation resistance of undifferentiated cells, which includes measuring the following (i) and/or (ii).
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) Promoter methylation status of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 (2) At least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 It is evaluated that the differentiation resistance of undifferentiated cells is low when the expression level and/or promoter activity of The method according to (1), wherein the differentiation resistance of undifferentiated cells is evaluated to be high when promoter activity is low.
(3) When the expression level and/or promoter activity of DPP6 is high, the differentiation resistance of undifferentiated cells is evaluated to be high, and when the expression level and/or promoter activity of DPP6 is low, the differentiation resistance of undifferentiated cells is evaluated. The method described in (1), which evaluates that the quality is low.
(4) When the methylation state of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, and MIR886 is low (because the higher the methylation state, the lower the expression (=promoter activity)), The differentiation resistance of differentiated cells is evaluated to be low, and the methylation state of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, and MIR886 is high (because the higher the methylation state, the lower the expression) The method according to (1), wherein the undifferentiated cells are evaluated to have high differentiation resistance.
(5) When the methylation state of the DPP6 promoter is low, the differentiation resistance of undifferentiated cells is evaluated to be high, and when the methylation state of the DPP6 promoter is high, the differentiation resistance of undifferentiated cells is evaluated to be low. Evaluation method described in (1).
(6) The undifferentiated cells are embryonic tumor cells (EC cells), embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), or embryonic germ cells (EG cells) (1) to ( 5) The method described in any one of 5).
(7) The method according to any one of (1) to (3) or (6), wherein the gene expression level is measured as the amount of mRNA or the amount of protein.
(8) The method of measuring the gene expression level is qPCR, digital PCR, immunostaining, in situ hybridization, RNA sequencing, microarray, NanoString, antibody array, FlowCytometry, mass spectrometry, or a combination thereof. Method.
(9) Methods for measuring the methylation status of gene promoters include concentrating methylated DNA and then detecting the concentrated DNA, and deciphering (sequencing) the base sequence after base substitution by bisulfite treatment. Methods: Detection of base sequences after base substitution by bisulfite treatment by hybridization; Methylation-specific PCR (MSP) method; Detection of cleavage with methylation-sensitive restriction enzymes; Detection of methylated cytosine The method according to any one of (1), (4) to (6), which is a method of glucosylating and detecting with an enzyme sensitive to glucosylated cytosine, or a combination thereof.
(10) A method for selecting undifferentiated cell lines with low or high differentiation resistance by measuring the following (i) and/or (ii) in an undifferentiated cell population.
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) Promoter methylation status of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 (11) At least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 When the expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is selected as an undifferentiated cell line with low differentiation resistance, the expression level and/or The method according to (10), which selects as an undifferentiated cell line with high differentiation resistance when the promoter activity is low.
(12) When the expression level and/or promoter activity of DPP6 is high, it is selected as an undifferentiated cell line with high differentiation resistance, and when the expression level and/or promoter activity of DPP6 is low, it is selected as an undifferentiated cell line with low differentiation resistance. The method described in (10) for selecting as a differentiated cell line.
(13) When the methylation state of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, and MIR886 is low, the cell line is selected as an undifferentiated cell line with low differentiation resistance, and ZNF354C, C12orf56, ZNF578 and MIR886, in which the methylation state of the promoter of at least one gene selected from the group consisting of MIR886 is high, the method is selected as an undifferentiated cell line with high differentiation resistance (10).
(14) When the methylation state of the DPP6 promoter is low, it is selected as an undifferentiated cell line with high differentiation resistance, and when the methylation state of the DPP6 promoter is high, it is selected as an undifferentiated cell line with low differentiation resistance. The method described in (10) for sorting.
(15) The undifferentiated cell line is an embryonic tumor cell (EC cell) line, an embryonic stem cell (ES cell) line, an induced pluripotent stem cell (iPS cell) line, or an embryonic germ cell (EG cell) line. The method according to any one of (10) to (14).
(16) Mark at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6, and MIR886 in order to detect undifferentiated cells with low or high resistance to differentiation that are present in the undifferentiated cell population. How to use it as.
(17) A kit for evaluating differentiation resistance of undifferentiated cells, comprising a reagent capable of measuring (i) and/or (ii) below.
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6, and MIR886 (18) The reagent capable of measuring the expression level of the gene is a primer, probe, or antibody. (17) The kit described.
(19) The kit according to (17), wherein the reagent capable of measuring gene promoter activity is a gene sequence in which a reporter protein is linked downstream of the promoter, or a vector incorporating this gene sequence.
(20) Reagents that can measure the methylation status of gene promoters include bisulfite (bisulfite), microarray reagent for methylation analysis, sequencing reagent by Sanger method, sequencing reagent for next-generation sequencer, 5-mC antibody, 5-hmC antibody, methyladenosine antibody, 5'-methyl-2'-deoxycytidine antibody, HRP-labeled DNA antibody, 5-hmC glucosyltransferase, glucosyl-5hmC-sensitive restriction enzyme endonuclease, MBD1 (Methyl-CpG Binding Domain Protein 1), The kit according to (17), which is MBD2 (Methyl-CpG Binding Domain Protein 2), a specific PCR primer, a specific probe, or a DNA purification kit.
本発明により、未分化細胞（例えば、未分化多能性幹細胞）の良否の早期判定による製造コストの抑制および未分化細胞から形成されるオルガノイド（例えば、肝臓器官芽(iPS細胞肝芽など)）の安全性の確保が達成される。
本明細書は、本願の優先権の基礎である日本国特許出願、特願2019‐005892の明細書および／または図面に記載される内容を包含する。According to the present invention, manufacturing costs can be reduced by early determination of the quality of undifferentiated cells (e.g., undifferentiated pluripotent stem cells), and organoids formed from undifferentiated cells (e.g., liver organ buds (iPS cell liver buds, etc.)) Safety is achieved.
This specification includes the contents described in the specification and/or drawings of the Japanese patent application, Japanese Patent Application No. 2019-005892, which is the basis of the priority of this application.
以下、本発明を詳細に説明する。 The present invention will be explained in detail below.
本発明は、下記の(i)及び／又は(ii)を測定することを含む、未分化細胞の分化抵抗性を評価する方法を提供する。
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態
発現レベル、プロモーター活性及びプロモーターのメチル化状態を測定する遺伝子は、１種類であってもよいし、２種類以上の組み合わせでもよい。The present invention provides a method for evaluating differentiation resistance of undifferentiated cells, which includes measuring (i) and/or (ii) below.
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) Promoter methylation status of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6, and MIR886 The expression level, promoter activity, and promoter methylation status are measured for only one gene. or a combination of two or more types.
評価の対象とする未分化細胞は、多能性を有する細胞であるとよく、例えば、未分化細胞は、胚性腫瘍細胞（ＥＣ細胞）、胚性幹細胞（ＥＳ細胞）又は人工多能性幹細胞（ｉＰＳ細胞）、胚性生殖細胞(EG細胞)である。未分化細胞は、ヒトあるいはヒト以外のいかなる動物に由来するものであってもよい。 The undifferentiated cells to be evaluated are preferably pluripotent cells. For example, the undifferentiated cells include embryonic tumor cells (EC cells), embryonic stem cells (ES cells), or induced pluripotent stem cells. (iPS cells) and embryonic germ cells (EG cells). The undifferentiated cells may be derived from humans or any non-human animal.
本明細書において、「分化抵抗性」とは、分化しにくさをいい、肝細胞への分化しにくさであることが好ましい。
未分化細胞の分化抵抗性は、細胞株によって異なることが知られているが、継代回数、継代方法や継代時の細胞密度などの培養条件によって、変化する可能性がある。あるいは培養用基質や培地によっても変化する可能性がある。
分化抵抗性は、分化誘導後に一定の細胞数の分化細胞を再播種し、未分化培養条件下で培養し、増殖したコロニーを未分化マーカー(SOX2など)による免疫染色で、未分化iPS細胞であることを確認し、1コロニーを1個の未分化iPS細胞であるとして算出することで評価できる。As used herein, "differentiation resistance" refers to difficulty in differentiation, and preferably difficulty in differentiation into hepatocytes.
It is known that the differentiation resistance of undifferentiated cells varies depending on the cell line, and may vary depending on culture conditions such as the number of passages, the passage method, and the cell density at the time of passage. Alternatively, it may change depending on the culture substrate or medium.
Differentiation resistance is determined by reseeding a certain number of differentiated cells after induction of differentiation, culturing under undifferentiated culture conditions, and immunostaining the proliferated colonies with undifferentiated markers (SOX2, etc.). It can be evaluated by confirming that there is one, and calculating that one colony is one undifferentiated iPS cell.
ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が高い場合に、未分化細胞の分化抵抗性が低いと評価し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が低い場合に、未分化細胞の分化抵抗性が高いと評価することができる。 When the expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, and MIR886 is high, it is evaluated that the differentiation resistance of undifferentiated cells is low. When the expression level and/or promoter activity of at least one gene selected from the group consisting of MIR886 is low, it can be evaluated that the undifferentiated cell has high differentiation resistance.
DPP6の発現レベル及び／又はプロモーター活性が高い場合に、未分化細胞の分化抵抗性が高いと評価し、DPP6の発現レベル及び／又はプロモーター活性が低い場合に、未分化細胞の分化抵抗性が低いと評価することができる。 When the expression level and/or promoter activity of DPP6 is high, the differentiation resistance of undifferentiated cells is evaluated to be high, and when the expression level and/or promoter activity of DPP6 is low, the differentiation resistance of undifferentiated cells is evaluated to be low. It can be evaluated as follows.
ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が低い場合に、未分化細胞の分化抵抗性が低いと評価し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が高い場合に、未分化細胞の分化抵抗性が高いと評価することができる。 When the methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, and MIR886 is low, the differentiation resistance of undifferentiated cells is evaluated to be low, and When the methylation state of the promoter of at least one gene selected from the group consisting of:
DPP6のプロモーターのメチル化状態が低い場合に、未分化細胞の分化抵抗性が高いと評価し、DPP6のプロモーターのメチル化状態が高い場合に、未分化細胞の分化抵抗性が低いと評価することができる。 When the methylation status of the DPP6 promoter is low, the differentiation resistance of undifferentiated cells is evaluated to be high, and when the methylation status of the DPP6 promoter is high, the differentiation resistance of undifferentiated cells is evaluated to be low. I can do it.
本明細書において、遺伝子の発現レベルが「高い」あるいは「低い」とは、未分化細胞の分化抵抗性の高いものと低いものを判別できる所定の数値よりも高いあるいは低いことを意味する。その数値は、求める感度や特異度によって異なるものであり、また、未分化細胞の種類（例えば、iPS細胞かES細胞かといった違い）、細胞のクローンの違いなどによって変化しうる。遺伝子のプロモーター活性が「高い」あるいは「低い」、メチル化状態の「高い」あるいは「低い」も同様である。 As used herein, the expression level of a gene is "high" or "low" means that it is higher or lower than a predetermined value that can be used to discriminate between undifferentiated cells with high differentiation resistance and low differentiation resistance. The numerical value differs depending on the desired sensitivity and specificity, and can also change depending on the type of undifferentiated cells (for example, iPS cells or ES cells), differences in cell clones, etc. The same applies to "high" or "low" gene promoter activity and "high" or "low" methylation status.
後述の実施例での実験によれば、遺伝子の発現レベルをqPCRで測定した場合、ZNF354Cの発現レベルの値が18Sを内部標準としたとき2x10^-7以上である場合に、未分化細胞の分化抵抗性が低いと評価でき、ZNF354Cの発現レベルの値が18Sを内部標準としたとき1x10^-7以下又は未満である場合に、未分化細胞の分化抵抗性が高いと評価できる。同じ測定法によれば、C12orf56の発現レベルの値が18Sを内部標準としたとき2.5x10^-7以上、ZNF578の発現レベルの値が18Sを内部標準としたとき1x10^-7以上、DPP6の発現レベルの値が18Sを内部標準としたとき3x10^-7以下又は未満、MIR886の発現レベルの値が18Sを内部標準としたとき1x10^-6以上である場合に、未分化細胞の分化抵抗性が低いと評価でき、C12orf56の発現レベルの値が18Sを内部標準としたとき2x10^-7以下又は未満、ZNF578の発現レベルの値が18Sを内部標準としたとき5x10^-8以下又は未満、DPP6の発現レベルの値が18Sを内部標準としたとき3x10^-7以上、MIR886の発現レベルの値が18Sを内部標準としたとき1x10^-7以下又は未満である場合に、未分化細胞の分化抵抗性が高いと評価できる。 According to the experiment in the example below, when the gene expression level is measured by qPCR, when the expression level value of ZNF354C is 2x10^-7 or more when 18S is used as an internal standard, it is determined that undifferentiated cells are It can be evaluated that the differentiation resistance of undifferentiated cells is low, and when the expression level of ZNF354C is less than or equal to 1x10^-7 when 18S is used as an internal standard, it can be evaluated that the differentiation resistance of undifferentiated cells is high. According to the same measurement method, the expression level of C12orf56 was 2.5x10^-7 or more when 18S was used as the internal standard, the expression level of ZNF578 was 1x10^-7 or more when 18S was used as the internal standard, and the expression level of DPP6 was 1x10^-7 or more when 18S was used as the internal standard. If the expression level value is less than or equal to 3x10^-7 when 18S is used as an internal standard, and the expression level value of MIR886 is 1x10^-6 or more when 18S is used as an internal standard, the differentiation resistance of undifferentiated cells is determined. The expression level of C12orf56 is less than or equal to 2x10^-7 when using 18S as the internal standard, and the expression level of ZNF578 is less than or equal to 5x10^-8 when using 18S as the internal standard. , undifferentiated cells when the expression level of DPP6 is 3x10^-7 or more when 18S is the internal standard, and the expression level of MIR886 is 1x10^-7 or less when 18S is the internal standard. can be evaluated as having high differentiation resistance.
また、後述の実施例での実験によれば、バイサルファイト処理実施後のイルミナ社Infinium MethylationEPIC BEadChip Kitを用いて遺伝子のプロモーターのメチル化状態を測定した場合、ZNF354Cのプロモーターのメチル化状態の値が40%以下又は未満である場合に、未分化細胞の分化抵抗性が低いと評価し、ZNF354Cのプロモーターのメチル化状態の値が60%以上である場合に、未分化細胞の分化抵抗性が高いと評価することができる。同じ測定法によれば、C12orf56のプロモーターのメチル化状態の値が40%以下又は未満、ZNF578のプロモーターのメチル化状態の値が40%以下又は未満、DPP6のプロモーターのメチル化状態の値が60%以上、MIR886のプロモーターのメチル化状態の値が40%以下又は未満、である場合に、未分化細胞の分化抵抗性が低いと評価し、C12orf56のプロモーターのメチル化状態の値が60%以上、ZNF578のプロモーターのメチル化状態の値が60%以上、DPP6のプロモーターのメチル化状態の値が40%以下又は未満、MIR886のプロモーターのメチル化状態の値が60%以上である場合に、未分化細胞の分化抵抗性が高いと評価することができる。 In addition, according to the experiment in the example below, when the methylation state of the gene promoter was measured using Illumina's Infinium MethylationEPIC BEadChip Kit after bisulfite treatment, the value of the methylation state of the promoter of ZNF354C was If the value is 40% or less, the differentiation resistance of undifferentiated cells is evaluated to be low, and if the value of the promoter methylation status of ZNF354C is 60% or more, the differentiation resistance of undifferentiated cells is evaluated to be high. It can be evaluated as follows. According to the same measurement method, the value of the promoter methylation status of C12orf56 is less than or equal to 40%, the value of the promoter methylation status of ZNF578 is less than or equal to 40%, and the value of the promoter methylation status of DPP6 is less than or equal to 40%. % or more, and the value of the promoter methylation state of MIR886 is 40% or less, the differentiation resistance of undifferentiated cells is evaluated to be low, and the value of the promoter methylation state of C12orf56 is 60% or more. , the value of the promoter methylation state of ZNF578 is 60% or more, the value of the promoter methylation state of DPP6 is 40% or less, and the value of the promoter methylation state of MIR886 is 60% or more. It can be evaluated that the differentiated cells have high differentiation resistance.
遺伝子の発現レベルは、遺伝子から転写されたmRNAの量あるいはmRNAから翻訳されたタンパク質の量として測定することができる。具体的には、遺伝子の発現レベルは、qPCR、デジタルPCR、免疫染色、in situ hybridization、RNAシークエンス、マイクロアレイ、NanoString、抗体アレイ、FlowCytometry、質量分析、それらの組み合わせなどで測定することができる。 The expression level of a gene can be measured as the amount of mRNA transcribed from the gene or the amount of protein translated from mRNA. Specifically, the gene expression level can be measured by qPCR, digital PCR, immunostaining, in situ hybridization, RNA sequencing, microarray, NanoString, antibody array, FlowCytometry, mass spectrometry, a combination thereof, and the like.
遺伝子のプロモーターのメチル化状態を解析する手法としては、１． 抗メチル化シトシンやアデノシン抗体などを用いてメチル化 DNA を濃縮し、濃縮後にシークエンス、マイクロアレイ、qPCR等によって、濃縮されたDNAを検出、定量する方法、２． バイサルファイト処理による塩基置換した後、塩基配列を解読(シークエンス)あるいはマイクロアレイ、MLPAR（Multiplex Ligation Probe Amplification）法等のハイブリダイズを用いて検出する方法、３．バイサルファイト処理による塩基置換した後、PCRによる増幅の有無で検出する方法（メチル化特異的PCR(MSP)法）、４． メチル化感受性の制限酵素を利用して切断の有無で検出する方法、５．メチル化シトシンをグルコシル化し、グルコシル化シトシン感受性の酵素で検出する方法等があり、これらのいずれかの方法あるいはその組み合わせなどを用いることができる。 Methods for analyzing the methylation status of gene promoters include 1. 2. A method of concentrating methylated DNA using anti-methylated cytosine or adenosine antibodies, etc., and detecting and quantifying the concentrated DNA by sequencing, microarray, qPCR, etc. after concentration; 2. 3. A method of detecting bases by deciphering the base sequence or hybridization such as microarray or MLPAR (Multiplex Ligation Probe Amplification) method after base substitution by bisulfite treatment; 3. 4. A method of detecting the presence or absence of amplification by PCR after base substitution by bisulfite treatment (methylation-specific PCR (MSP) method); 5. A method of detecting the presence or absence of cleavage using a methylation-sensitive restriction enzyme; 5. There are methods such as glucosylating methylated cytosine and detecting it with an enzyme sensitive to glucosylated cytosine, and any of these methods or a combination thereof can be used.
未分化細胞集団における、上記の(i)及び／又は(ii)を測定することによって、分化抵抗性の低いあるいは高い未分化細胞株を選別することができる。よって、本発明は、未分化細胞集団における、下記の(i)及び／又は(ii)を測定することによって、分化抵抗性の低いあるいは高い未分化細胞株を選別する方法を提供する。
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態
発現レベル、プロモーター活性及びプロモーターのメチル化状態を測定する遺伝子は、１種類であってもよいし、２種類以上の組み合わせでもよい。By measuring (i) and/or (ii) above in an undifferentiated cell population, undifferentiated cell lines with low or high differentiation resistance can be selected. Therefore, the present invention provides a method for selecting undifferentiated cell lines with low or high differentiation resistance by measuring the following (i) and/or (ii) in an undifferentiated cell population.
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) Promoter methylation status of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6, and MIR886 The expression level, promoter activity, and promoter methylation status are measured for only one gene. or a combination of two or more types.
選別の対象とする未分化細胞株は、多能性を有する細胞株であるとよく、例えば、未分化細胞株は、胚性腫瘍細胞（ＥＣ細胞）株、胚性幹細胞（ＥＳ細胞）株、人工多能性幹細胞（ｉＰＳ細胞）株又は胚性生殖細胞（ＥＧ細胞)株であり、ｉＰＳ細胞株が好ましい。未分化細胞株は、ヒトあるいはヒト以外のいかなる動物に由来するものであってもよい。 The undifferentiated cell line to be selected is preferably a pluripotent cell line. For example, the undifferentiated cell line may include an embryonic tumor cell (EC cell) line, an embryonic stem cell (ES cell) line, An induced pluripotent stem cell (iPS cell) line or an embryonic germ cell (EG cell) line, with iPS cell lines being preferred. The undifferentiated cell line may be derived from a human or any non-human animal.
ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が高い場合に、分化抵抗性が低い未分化細胞株として選別し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が低い場合に、分化抵抗性が高い未分化細胞株として選別することができる。 When the expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, and MIR886 is high, the cell line is selected as an undifferentiated cell line with low differentiation resistance. When the expression level and/or promoter activity of at least one gene selected from the group consisting of MIR886 is low, the cell line can be selected as an undifferentiated cell line with high differentiation resistance.
DPP6の発現レベル及び／又はプロモーター活性が高い場合に、分化抵抗性が高い未分化細胞株として選別し、DPP6の発現レベル及び／又はプロモーター活性が低い場合に、分化抵抗性が低い未分化細胞株として選別することができる。 When the expression level and/or promoter activity of DPP6 is high, it is selected as an undifferentiated cell line with high differentiation resistance, and when the expression level and/or promoter activity of DPP6 is low, it is selected as an undifferentiated cell line with low differentiation resistance. It can be selected as
ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が低い場合に、分化抵抗性が低い未分化細胞株として選別し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が高い場合に、分化抵抗性が高い未分化細胞株として選別することができる。 When the methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, and MIR886 is low, the cell line is selected as an undifferentiated cell line with low differentiation resistance. When the methylation state of the promoter of at least one gene selected from the group consisting of:
DPP6のプロモーターのメチル化状態が低い場合に、分化抵抗性が高い未分化細胞株として選別し、DPP6のプロモーターのメチル化状態が高い場合に、分化抵抗性が低い未分化細胞株として選別することができる。 When the methylation state of the DPP6 promoter is low, it is selected as an undifferentiated cell line with high differentiation resistance, and when the methylation state of the DPP6 promoter is high, it is selected as an undifferentiated cell line with low differentiation resistance. Can be done.
遺伝子の発現レベル、プロモーター活性及び遺伝子のプロモーターのメチル化状態の「高い」あるいは「低い」については上述した通りである。 The terms "high" and "low" in the gene expression level, promoter activity, and methylation state of the gene promoter are as described above.
ZNF354C、C12orf56、ZNF578、DPP6及びMIR886は、未分化細胞集団中に存在する、分化抵抗性の低いあるいは高い未分化細胞を検出するためのマーカー遺伝子として使用できる。よって、本発明は、未分化細胞集団中に存在する、分化抵抗性の低いあるいは高い未分化細胞を検出するために、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子をマーカーとして使用する方法を提供する。
マーカーとして使用する遺伝子は、１種類であってもよいし、２種類以上の組み合わせでもよい。ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 can be used as marker genes for detecting undifferentiated cells with low or high differentiation resistance present in the undifferentiated cell population. Therefore, the present invention provides at least one cell selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6, and MIR886 in order to detect undifferentiated cells with low or high resistance to differentiation that are present in an undifferentiated cell population. A method of using genes as markers is provided.
One type of gene may be used as a marker, or a combination of two or more types may be used.
また、本発明は、下記の(i)及び／又は(ii)を測定可能な試薬を含む、未分化細胞の分化抵抗性を評価するためのキットを提供する。
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態
発現レベル、プロモーター活性及びプロモーターのメチル化状態を測定する遺伝子は、１種類であってもよいし、２種類以上の組み合わせでもよい。The present invention also provides a kit for evaluating differentiation resistance of undifferentiated cells, which includes a reagent capable of measuring (i) and/or (ii) below.
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) Promoter methylation status of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6, and MIR886 The expression level, promoter activity, and promoter methylation status are measured for only one gene. or a combination of two or more types.
遺伝子の発現レベルを測定可能な試薬としては、プライマー、プローブ及び抗体などを挙げることができ、例えば、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の転写産物（mRNA）又はcDNAを特異的に増幅できるオリゴヌクレオチドプライマーのセット、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の転写産物（mRNA）又はcDNAに特異的にハイブリダイズするヌクレオチドプローブ、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の転写産物（mRNA）から翻訳されたタンパク（翻訳産物）に特異的に結合する抗体である。オリゴヌクレオチドプライマーのセットは、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の転写産物（mRNA）又はcDNAのヌクレオチド配列中の標的配列（通常、５０～１８０ｂｐ程度）を増幅できるものであるとよく、標的配列の両末端と相補的な配列を有するように設計されるとよい。オリゴヌクレオチドプライマーの長さは、例えば、１５～３５ヌクレオチドであるとよく、１８～２７ヌクレオチドが好ましい。ヌクレオチドプローブは、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の転写産物（mRNA）又はcDNAにストリンジェントな条件下でハイブリダイズするものであるとよく、前記mRNA又はcDNAのヌクレオチド配列の一部又は全部又はそれに相補的な配列を有するように設計されるとよい。ストリンジェントな条件は適宜決定することができる。ヌクレオチドプローブの長さは、通常１０００ヌクレオチド以下、好ましくは、１００ヌクレオチド以下、より好ましくは５０ヌクレオチド以下、さらにより好ましくは、１４～３０ヌクレオチドである。ヌクレオチドプローブは、一本鎖であっても、二本鎖であってもよい。抗体は、モノクローナル抗体又はポリクローナル抗体のいずれであってもよい。本明細書において、抗体とは、全長抗体の他、Fab、F(ab)’2、ScFv、Diabody、VH、VL、Sc(Fv)2、Bispecific sc(Fv)2、Minibody、ScFv-Fc monomer、ScFv-Fc dimerなどの低分子化されたものも含む概念である。プローブや抗体は、固相（例えば、基板、ビーズ、膜など）上に固定されていてもよい。Examples of reagents that can measure the expression level of a gene include primers, probes, and antibodies. A set of oligonucleotide primers capable of specifically amplifying mRNA) or cDNA, which specifically hybridizes to the transcript (mRNA) or cDNA of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6, and MIR886. This is an antibody that specifically binds to a protein (translation product) translated from a transcription product (mRNA) of at least one gene selected from the group consisting of a nucleotide probe, ZNF354C, C12orf56, ZNF578, DPP6, and MIR886. The set of oligonucleotide primers is a target sequence (usually about 50 to 180 bp) in the nucleotide sequence of the transcript (mRNA) or cDNA of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6, and MIR886. It is preferable that the target sequence can be amplified, and it is preferable that it is designed to have sequences complementary to both ends of the target sequence. The length of the oligonucleotide primer may be, for example, 15 to 35 nucleotides, preferably 18 to 27 nucleotides. The nucleotide probe is preferably one that hybridizes under stringent conditions to the transcript (mRNA) or cDNA of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6, and MIR886, and the Alternatively, it may be designed to have part or all of the nucleotide sequence of cDNA or a sequence complementary thereto. Stringent conditions can be determined as appropriate. The length of the nucleotide probe is usually 1000 nucleotides or less, preferably 100 nucleotides or less, more preferably 50 nucleotides or less, and even more preferably 14 to 30 nucleotides. Nucleotide probes may be single-stranded or double-stranded. The antibody may be either a monoclonal antibody or a polyclonal antibody. In this specification, antibodies include full-length antibodies, Fab, F(ab)' 2 , ScFv, Diabody, V H , V L , Sc(Fv) 2 , Bispecific sc(Fv) 2 , Minibody, ScFv- This concept also includes lower molecular weight products such as Fc monomer and ScFv-Fc dimer. Probes and antibodies may be immobilized on a solid phase (eg, a substrate, beads, membrane, etc.).
本発明の試薬は標識されてもよい。例えば、プライマーは、蛍光物質や消光物質などにより標識されていてもよく、プローブ及び抗体は、放射性同位元素、酵素、発光物質、蛍光物質、ビオチンなどで標識されてもよい。また、ターゲット分子（本発明では、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現産物であるタンパク質）に特異的に結合する一次抗体の反応後、この一次抗体に結合する二次抗体を反応させて、ターゲット分子の検出を行う場合には、二次抗体を標識するとよい（一次抗体は標識しない）。 Reagents of the invention may be labeled. For example, primers may be labeled with fluorescent substances, quenching substances, etc., and probes and antibodies may be labeled with radioisotopes, enzymes, luminescent substances, fluorescent substances, biotin, etc. In addition, after reaction with a primary antibody that specifically binds to a target molecule (in the present invention, a protein that is an expression product of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6, and MIR886), this primary When detecting a target molecule by reacting a secondary antibody that binds to an antibody, it is preferable to label the secondary antibody (the primary antibody is not labeled).
遺伝子のプロモーター活性を測定可能な試薬としては、プロモーター下流にレポータータンパク質を連結した遺伝子配列又はこの遺伝子配列を組み込んだベクターなどを挙げることができる。レポータータンパク質としては、ルシフェラーゼ、GFPなどの蛍光タンパク質、CD抗原などの細胞膜に発現するタンパク質などを例示することができる。ベクターはプラスミドベクターが好ましい。 Examples of reagents that can measure gene promoter activity include a gene sequence in which a reporter protein is linked downstream of the promoter, or a vector incorporating this gene sequence. Examples of reporter proteins include fluorescent proteins such as luciferase and GFP, and proteins expressed on cell membranes such as CD antigen. The vector is preferably a plasmid vector.
遺伝子のプロモーターのメチル化状態を測定可能な試薬としては、バイサルファイト（亜硫酸水素塩）、Illumina 社 Infinium MethylationEPIC BeadChip等のメチル化解析用マイクロアレイ試薬、Sanger法によるシークエンス試薬、次世代シークエンサー用シークエンス試薬、5-mC抗体、5-hmC抗体、メチルアデノシン抗体、5’-methyl-2’-deoxycytidine抗体、HRP標識DNA抗体、5-hmC グルコシルトランスフェラーゼ、グルコシル-5hmC感受性制限酵素エンドヌクレアーゼ（GSRE: MspI、GlaI、Csp6I、HaeIII、TaqαI、MboI、McrBC)、MBD1 (Methyl-CpG Binding Domain Protein1)、MBD2 (Methyl-CpG Binding Domain Protein2)、特異的PCRプライマー、特異的プローブ又はDNA精製キットなどを例示することができる。 Reagents that can measure the methylation status of gene promoters include bisulfite (bisulfite), microarray reagents for methylation analysis such as Illumina's Infinium MethylationEPIC BeadChip, sequencing reagents using the Sanger method, sequencing reagents for next-generation sequencers, 5-mC antibody, 5-hmC antibody, methyladenosine antibody, 5'-methyl-2'-deoxycytidine antibody, HRP-labeled DNA antibody, 5-hmC glucosyltransferase, glucosyl-5hmC sensitive restriction enzyme endonuclease (GSRE: MspI, GlaI , Csp6I, HaeIII, TaqαI, MboI, McrBC), MBD1 (Methyl-CpG Binding Domain Protein 1), MBD2 (Methyl-CpG Binding Domain Protein 2), specific PCR primers, specific probes, or DNA purification kits. can.
本発明のキットは、さらに、プライマーで検出するための試薬（ＤＮＡポリメラーゼ、バッファー、マグネシウムイオン、ｄＮＴＰｓ、プローブなど）、プローブで検出するための試薬（バッファー、抗体、基質など）、抗体で検出するための試薬（二次抗体、基質、バッファーなど）、遺伝子のプロモーター活性を測定するための試薬（バッファー、発光基質、抗体など）、器具（反応容器、ピペットなど）、キットの使用説明書、対照用の試料、測定結果を解析するための対照データなどを含んでもよい。 The kit of the present invention further includes reagents for detection with primers (DNA polymerase, buffer, magnesium ions, dNTPs, probes, etc.), reagents for detection with probes (buffers, antibodies, substrates, etc.), and detection with antibodies. reagents for measuring gene promoter activity (secondary antibodies, substrates, buffers, etc.), reagents for measuring gene promoter activity (buffers, luminescent substrates, antibodies, etc.), equipment (reaction vessels, pipettes, etc.), kit instructions, controls. It may also include a sample for use, control data for analyzing measurement results, etc.
本発明のマーカー遺伝子（ZNF354C、C12orf56、ZNF578、DPP6及びMIR886）は以下のようにして見出された。継代回数が異なる同一iPS細胞クローンの分化細胞における未分化iPS細胞残存量を評価し、未分化iPS細胞が残存するiPS細胞と残存しないiPS細胞におけるDNAメチル化解析を実施し、DNAメチル化状態に差が有る遺伝子について、分化させる前のiPS細胞時点での遺伝子発現を調べ、未分化残存し易いクローンと未分化残存し難いクローンの間で発現に相違のある遺伝子を同定する。同定された遺伝子がマーカー遺伝子の候補となる。よって、本発明は、継代回数などの培養状態が異なる同一未分化細胞クローンの分化細胞における未分化細胞残存量を評価し、未分化細胞が残存するクローンと未分化細胞が残存しないクローンにおけるDNAメチル化解析を実施し、DNAメチル化状態に差が有る遺伝子について、分化させる前の未分化細胞で遺伝子発現を調べ、未分化のまま残存し易いクローンと未分化のまま残存し難いクローンの間で発現に相違のある遺伝子を同定することを含む、未分化細胞集団中に存在する、分化抵抗性の低いあるいは高い未分化細胞を検出するためのマーカー遺伝子を探索する方法を提供する。"DNAメチル化状態に差が有る遺伝子"が他のクローンなどでも共通して、未分化細胞の残存と相関が有れば、遺伝子発現ではなくメチル化状態を評価することによって予測することが出来るマーカーとなる。 The marker genes of the present invention (ZNF354C, C12orf56, ZNF578, DPP6 and MIR886) were discovered as follows. We evaluated the remaining amount of undifferentiated iPS cells in differentiated cells of the same iPS cell clone with different passage numbers, performed DNA methylation analysis on iPS cells with and without remaining undifferentiated iPS cells, and determined the DNA methylation status. For genes with differences in expression, we will examine the gene expression at the time of iPS cells before differentiation, and identify genes with differences in expression between clones that tend to remain undifferentiated and clones that are difficult to remain undifferentiated. The identified genes become candidates for marker genes. Therefore, the present invention evaluates the remaining amount of undifferentiated cells in differentiated cells of the same undifferentiated cell clones with different culture conditions such as the number of passages, and determines the amount of DNA in the clones in which undifferentiated cells remain and in the clones in which no undifferentiated cells remain. Methylation analysis is performed to examine gene expression in undifferentiated cells before differentiation for genes with differences in DNA methylation status, and to determine the difference between clones that tend to remain undifferentiated and clones that are difficult to remain undifferentiated. The present invention provides a method for searching for marker genes for detecting undifferentiated cells with low or high resistance to differentiation, which are present in an undifferentiated cell population, and includes identifying genes differentially expressed in a population of undifferentiated cells. If "genes with differences in DNA methylation status" are common among other clones and are correlated with the survival of undifferentiated cells, predictions can be made by evaluating the methylation status rather than gene expression. Becomes a marker.
未分化細胞は、多能性を有する細胞であるとよく、例えば、未分化細胞は、胚性腫瘍細胞（ＥＣ細胞）、胚性幹細胞（ＥＳ細胞）又は人工多能性幹細胞（ｉＰＳ細胞）、胚性生殖細胞(EG細胞)である。未分化細胞は、ヒトあるいはヒト以外のいかなる動物に由来するものであってもよい。 The undifferentiated cells are preferably pluripotent cells, for example, the undifferentiated cells include embryonic tumor cells (EC cells), embryonic stem cells (ES cells), or induced pluripotent stem cells (iPS cells), They are embryonic germ cells (EG cells). The undifferentiated cells may be derived from humans or any non-human animal.
培養状態としては、継代回数、継代方法や継代時の細胞密度などの培養条件、培養用基質や培地などを例示することができる。
未分化細胞の継代回数は、少なくとも１回以上であればよく、未分化細胞の分化抵抗性が高い場合の継代数は、４０～１００が好ましく、未分化細胞の分化抵抗性が低い場合の継代数は８～３０が好ましい。iPS細胞は無限に増殖すると考えられているので継代数は何回でも無限でも可能である。Examples of the culture state include the number of passages, culture conditions such as the passage method and cell density at the time of passage, and the culture substrate and medium.
The number of passages of undifferentiated cells should be at least one or more, and the number of passages when undifferentiated cells have high resistance to differentiation is preferably 40 to 100, and the number of passages when undifferentiated cells have low resistance to differentiation is preferably 40 to 100. The passage number is preferably 8 to 30. Since iPS cells are thought to proliferate indefinitely, they can be passaged an infinite number of times.
分化細胞は、内胚葉、中胚葉及び外胚葉のいずれの分化細胞であってもよい。内胚葉の分化細胞としては、肝内胚葉細胞などを例示することができるが、これに限定されるわけではない。中胚葉の分化細胞としては、横中隔間充織細胞、間葉系細胞、血管内皮細胞などを例示することができるが、これらに限定されるわけではない。外胚葉の分化細胞としては、神経幹細胞、神経堤細胞、神経細胞などを例示することができるが、これに限定されるわけではない。 The differentiated cells may be endodermal, mesodermal, or ectodermal differentiated cells. Examples of endoderm differentiated cells include hepatic endoderm cells, but are not limited thereto. Examples of mesodermal differentiated cells include, but are not limited to, transverse septal mesenchymal cells, mesenchymal cells, and vascular endothelial cells. Examples of ectodermal differentiated cells include, but are not limited to, neural stem cells, neural crest cells, and nerve cells.
未分化細胞残存量の評価は後述の実施例に記載の方法で行うことができるが、これに限定されるわけではない
。The remaining amount of undifferentiated cells can be evaluated by the method described in Examples below, but is not limited thereto.
DNAメチル化解析、遺伝子発現の測定法は上述の通りである。 DNA methylation analysis and gene expression measurement methods were as described above.
以下、実施例により本発明を具体的に説明する。本発明の範囲は以下の実施例に限定されるものではない。
〔実施例１〕iPS細胞時点で未分化残存の評価が可能なマーカーの同定
目的
再生医療応用に資するiPS(ES)細胞由来分化細胞における未分化細胞の混入の検出および排除は、すべてのiPS(ES)細胞由来細胞加工製品の安全性の確保における重要な課題である。これまでに網膜色素上皮細胞(RPE)におけるLIN28Aの発現検証による迅速な未分化細胞の混入評価が報告されているが、我々は複数の細胞加工製品においてLIN28Aでの評価が不適であることを見出している。また、製造の最終段階における品質評価では、その時点で規格外となった場合に、時間および経済的に多大な損失が生じるという問題があり、製造の遅れは時に患者の予後をも左右することも考えられる。
iPS(ES)細胞由来細胞加工製品の安全性の確保のため、最終製品の迅速・超高感度な評価手法の開発が必要である。さらに時間的、経済的観点から製造のより早い段階すなわち、未分化iPS細胞の段階で未分化細胞が最終製品に残存する可能性を予測することが望まれる。
そこで本発明では未分化iPS細胞の段階で最終製品に残存する可能性を予測することが可能な評価手法を開発した。
これまでにiPS細胞から分化誘導した三胚葉いずれの分化細胞においても利用可能な、汎用性の高い未分化マーカーを同定している（「未分化細胞検出法」(特願2018-115025))。この研究の中で継代回数が長いiPS細胞において未分化残存が起こりやすいクローンを同定している。さらに、この未分化残存の起こりやすさはジェネティックな変異（遺伝子変異）ではなく、エピジェティックな修飾であることを強く示唆するデータを得た。そこで、未分化残存が起こりやすいiPS細胞クローンと未分化残存が起こりにくいiPS細胞クローンのDNAメチル化状態に違いがある遺伝子を抽出し、さらに定量PCRにより発現と未分化残存に相関がある遺伝子(MIR886(VTRNA2-1)、DPP6、ZNF578、C12orf56、ZNF354C)を抽出した。Hereinafter, the present invention will be specifically explained with reference to Examples. The scope of the invention is not limited to the following examples.
[Example 1] Identification of markers that allow evaluation of undifferentiated status at the time of iPS cells
the purpose
Detection and elimination of contamination of undifferentiated cells in iPS (ES) cell-derived differentiated cells that contribute to regenerative medicine applications is an important issue in ensuring the safety of all iPS (ES) cell-derived cell processed products. Up to now, rapid evaluation of undifferentiated cell contamination by verifying the expression of LIN28A in retinal pigment epithelial cells (RPE) has been reported, but we found that evaluation using LIN28A is inappropriate for multiple cell processing products. ing. In addition, in quality evaluation at the final stage of manufacturing, there is a problem in that if it is out of specification at that point, it will cause a huge loss in terms of time and money, and delays in manufacturing can sometimes affect the prognosis of patients. can also be considered.
To ensure the safety of iPS(ES) cell-derived cell-processed products, it is necessary to develop rapid and ultra-sensitive evaluation methods for final products. Furthermore, from a time and economic perspective, it is desirable to predict the possibility that undifferentiated cells will remain in the final product at an earlier stage of manufacturing, ie, at the undifferentiated iPS cell stage.
Therefore, in the present invention, we have developed an evaluation method that can predict the possibility that undifferentiated iPS cells will remain in the final product at the stage of undifferentiated iPS cells.
So far, we have identified a highly versatile undifferentiated marker that can be used in differentiated cells of any of the three germ layers induced to differentiate from iPS cells (``Undifferentiated cell detection method'' (Patent application 2018-115025)). In this study, we identified clones that tend to remain undifferentiated in iPS cells that have been passaged for a long time. Furthermore, we obtained data that strongly suggest that the likelihood of this undifferentiated persistence is due to epigetic modification rather than genetic variation. Therefore, we extracted genes that have a difference in DNA methylation status between iPS cell clones that are more likely to remain undifferentiated and those that are less likely to remain undifferentiated. MIR886 (VTRNA2-1), DPP6, ZNF578, C12orf56, ZNF354C) were extracted.
方法
[分化抵抗性評価のための未分化残存試験]
ヒトiPS細胞から肝細胞を分化誘導し、iPS細胞肝芽の作製に最適なタイミングで胚体内胚葉（DE)の2-6日後(HEとする)にPLoS One. 2014 Oct 27;9(10):e110496に記載の方法を一部改変し、ディッシュコーティング剤としてLaminin511E8(iMatrix,ニッピ)、培養培地としてStemFitを用いて1週間培養し、出現したコロニーをSOX2, TRA1-60などの未分化iPS細胞マーカーで免疫染色することにより、出現したコロニーが未分化iPS細胞コロニーであることを確認する。未分化コロニー1つは分化細胞に含まれる未分化iPS細胞1細胞より形成されたコロニーとして、分化細胞に含まれる未分化iPS細胞の細胞数を評価する。
ヒトiPS細胞から肝細胞の分化誘導はNature. 499(7459):481-4.(2013); Cell Rep. 21(10):2661-2670.(2017)、に記載の通りに行った。
[メチル化解析]
メチル化解析は各分化段階の細胞のDNA について、バイサルファイト処理実施後、イルミナ社Infinium MethylationEPIC BeadChip Kit を用いて、DNA メチル化解析を行った。
[発現定量評価]
定量PCRはNature. 499(7459):481-4.(2013); Cell Rep. 21(10):2661-2670.(2017)、に記載の通りに行った。 Method
[Undifferentiated residual test for evaluation of differentiation resistance]
Differentiation of hepatocytes is induced from human iPS cells, and PLoS One. 2-6 days after definitive endoderm (DE) (HE) is the optimal timing for producing iPS cell liver buds. 2014 Oct 27;9(10) :e110496 was partially modified and cultured for one week using Laminin511E8 (iMatrix, Nippi) as a dish coating agent and StemFit as a culture medium, and the colonies that appeared were used as undifferentiated iPS cells such as SOX2 and TRA1-60. Confirm that the colonies that appear are undifferentiated iPS cell colonies by immunostaining with markers. One undifferentiated colony is a colony formed from one undifferentiated iPS cell included in the differentiated cells, and the number of undifferentiated iPS cells included in the differentiated cells is evaluated.
Differentiation of hepatocytes from human iPS cells was induced as described in Nature. 499(7459):481-4.(2013); Cell Rep. 21(10):2661-2670.(2017).
[Methylation analysis]
Methylation analysis was performed on the DNA of cells at each differentiation stage using Illumina's Infinium MethylationEPIC BeadChip Kit after bisulfite treatment.
[Expression quantitative evaluation]
Quantitative PCR was performed as described in Nature. 499(7459):481-4.(2013); Cell Rep. 21(10):2661-2670.(2017).
結果
各マーカー遺伝子の発現量と、再培養法にて評価した肝内胚葉細胞(HE)での残存未分化iPS細胞数の相関を図１に示す。未分化残存しやすい株では、再培養法で未分化iPS細胞の残存が見られ、未分化残存し難い株では、未分化iPS細胞の残存が検出されない。各マーカー遺伝子は未分化残存した時(未分化残存し難い株)と未分化残存しなかった時(未分化残存しやすい株)の間で明確な発現の違いが有る。すなわち、ZNF354C、C12orf56、ZNF578及びMIR886では、未分化残存が無い場合に、未分化残存が有る場合に比べ、遺伝子発現が高く、一方DPP6では、未分化残存が無い場合に、未分化残存が有る場合に比べ、遺伝子発現が低い。 Results Figure 1 shows the correlation between the expression level of each marker gene and the number of remaining undifferentiated iPS cells in hepatic endoderm cells (HE) evaluated by the re-culture method. In strains that tend to remain undifferentiated, residual undifferentiated iPS cells are observed by re-cultivation, and in strains that are difficult to remain undifferentiated, residual undifferentiated iPS cells are not detected. There is a clear difference in the expression of each marker gene between when it remains undifferentiated (strains that are unlikely to remain undifferentiated) and when it does not remain undifferentiated (strains that are likely to remain undifferentiated). That is, for ZNF354C, C12orf56, ZNF578, and MIR886, gene expression is higher in the absence of undifferentiated residuals than in the presence of undifferentiated residuals, whereas in DPP6, there is undifferentiated residuals in the absence of undifferentiated residuals. gene expression is lower than in the case of
まとめ
・iPS細胞はクローンの特性によるもの以外に、培養状態(継代回数など)によって、分化細胞に未分化iPS細胞が残存する場合が有る。
・継代回数の増加によって生じる未分化iPS細胞が残存しやすいiPS細胞はエピジェネティックな変化によって生じる。
・メチル化変異によって遺伝子発現が変化し、未分化iPS細胞の残存しやすさと相関する遺伝子を同定した。この遺伝子は、iPS細胞の段階で分化細胞における未分化iPS細胞の残存のしやすさを類推するマーカーとなる。
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 Summary : In addition to the characteristics of the clone, undifferentiated iPS cells may remain among differentiated cells depending on the culture conditions (number of passages, etc.).
・Undifferentiated iPS cells that are generated by increasing the number of passages tend to persist iPS cells are caused by epigenetic changes.
・We identified genes whose gene expression changes due to methylation mutations and which correlates with the likelihood of survival of undifferentiated iPS cells. This gene serves as a marker for estimating the ease with which undifferentiated iPS cells survive in differentiated cells at the iPS cell stage.
All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.
本発明は、再生医療に用いるiPS細胞などの未分化細胞の品質評価に利用できる。具体的には、iPS細胞の樹立工程と、マスターセルバンク、ワーキングセルバンクの製造工程、再生医療等製品の製造工程などにおける、未分化細胞の残存/混入の検出や評価に利用することができる。 The present invention can be used to evaluate the quality of undifferentiated cells such as iPS cells used in regenerative medicine. Specifically, it can be used to detect and evaluate residual/contamination of undifferentiated cells in the iPS cell establishment process, the manufacturing process of master cell banks and working cell banks, and the manufacturing process of regenerative medicine products.
Claims (20)
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態 A kit for evaluating resistance to differentiation of undifferentiated cells into hepatic endoderm cells, comprising a reagent capable of measuring (i) and/or (ii) below.
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
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