JP2018117582A - Method for evaluating quality of stem cell and kit for evaluating quality of stem cell - Google Patents
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Abstract
Description
本発明は、特定のタンパク質又はそれをコードする遺伝子の発現を指標として幹細胞の品質を評価する方法及び幹細胞の品質評価用キットに関する。 The present invention relates to a method for evaluating stem cell quality using the expression of a specific protein or a gene encoding the same as an index, and a stem cell quality evaluation kit.
哺乳動物の組織は、傷害若しくは疾患、又は加齢等に伴い細胞・臓器の損傷が起こった場合、再生系が働き、細胞・臓器の損傷を回復しようとする。この作用に、当該組織に備わる幹細胞が大きな役割を果たしている。幹細胞は、自分自身と同じ性質をもった細胞を産生する能力（自己複製能）と、機能する細胞へと分化する能力（分化能）を併せ持った未分化な細胞であり、この性質により細胞・臓器の損傷部を補うことで回復に導くと考えられている。近年、このような幹細胞を応用した、次世代の医療として再生医療に期待が高まっている。 In mammalian tissues, when cell or organ damage occurs due to injury or disease, or aging, the regeneration system works and tries to recover the damage of cells or organs. Stem cells provided in the tissue play a major role in this effect. Stem cells are undifferentiated cells that have both the ability to produce cells with the same properties as themselves (self-renewal ability) and the ability to differentiate into functional cells (differentiation ability). It is thought to lead to recovery by supplementing the damaged part of the organ. In recent years, there is an increasing expectation for regenerative medicine as next-generation medicine using such stem cells.
幹細胞を医療用又は研究用に利用する場合、その品質管理は非常に重要である。例えば、医療応用を想定するとき、細胞の品質は患者の安全性に直結するばかりか、治療効果の確実性や安定性にも大きく影響する。また、研究応用であっても、細胞の品質は実験・検証の成功や再現性に関わる。一方で、人体から採取された幹細胞は、不均一な細胞集団であり、その品質（分化能・増殖能・未分化性など）は、ドナー、培養条件、細胞継代数等により大きく変化する。したがって、近年、幹細胞の品質を評価する技術の確立が求められている。例えば、幹細胞の品質評価方法として、幹細胞の培養上清中に分泌されたｍｉＲＮＡを指標とする方法や（特許文献１）、特定のタンパク質（Ｗｎｔ５ａレセプターであるＦｚｄ５及びＲｏｒ２）の発現を指標とする方法（特許文献２）、特定遺伝子のＣｐＧアイランドのＤＮＡメチル化度を指標とする方法（特許文献３）等が報告されているが、いまだ十分満足できるものではなかった。 When stem cells are used for medical or research purposes, quality control is very important. For example, when assuming medical applications, cell quality not only directly affects patient safety, but also greatly affects the certainty and stability of therapeutic effects. Even in research applications, cell quality is related to the success and reproducibility of experiments and verifications. On the other hand, stem cells collected from the human body are heterogeneous cell populations, and their quality (differentiation ability / proliferation ability / undifferentiation property, etc.) varies greatly depending on donors, culture conditions, cell passage number, and the like. Therefore, in recent years, establishment of a technique for evaluating the quality of stem cells has been demanded. For example, as a quality evaluation method for stem cells, a method using miRNA secreted in the culture supernatant of stem cells as an index (Patent Document 1), or expression of specific proteins (Wz5a receptors Fzd5 and Ror2) is used as an index. Although a method (Patent Document 2), a method using the degree of DNA methylation of a CpG island of a specific gene as an index (Patent Document 3), and the like have been reported, they have not been fully satisfactory.
従って、本発明は、再生医療材料、再生美容材料、又は研究応用に用いる幹細胞の新たな品質評価方法を提供することを目的とする。 Therefore, an object of the present invention is to provide a new quality evaluation method for regenerative medical materials, regenerative cosmetic materials, or stem cells used for research applications.
本発明者らは、上記課題を解決するため鋭意研究を行った結果、ＧＲＥＭ１及び／又はＧＲＥＭ２の発現量が低い幹細胞は、各種細胞への分化能が高く、ＧＲＥＭ１及び／又はＧＲＥＭ２の発現量と幹細胞の分化能に負の相関関係があること、また、かかる相関関係に基づきＧＲＥＭ１及び／又はＧＲＥＭ２の発現を指標とすれば、幹細胞の品質、特にその分化能を評価できることを見出し、本発明を完成させるに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that stem cells with a low expression level of GREM1 and / or GREM2 have high differentiation potential into various cells, and the expression levels of GREM1 and / or GREM2 It has been found that there is a negative correlation with the differentiation potential of stem cells, and that the expression of GREM1 and / or GREM2 can be used as an index based on such correlation, and the quality of stem cells, particularly its differentiation potential, can be evaluated. It came to complete.
すなわち、本発明は、以下の発明を包含する。
（１）ＧＲＥＭ１タンパク質及び／又はＧＲＥＭ２タンパク質、又は、ＧＲＥＭ１タンパク質をコードする遺伝子及び／又はＧＲＥＭ２タンパク質をコードする遺伝子の発現量を指標として、哺乳動物の幹細胞の品質を評価する方法。
（２）前記幹細胞が間葉系幹細胞である（１）に記載の方法。
（３）前記品質が分化能である（１）又は（２）に記載の方法。
（４）ＧＲＥＭ１タンパク質及び／又はＧＲＥＭ２タンパク質、又は、ＧＲＥＭ１タンパク質をコードする遺伝子及び／又はＧＲＥＭ２タンパク質をコードする遺伝子に対して特異的親和性を有する物質を少なくとも１種含む、幹細胞の品質評価用キット。
（５）前記特異的親和性を有する物質が、ＧＲＥＭ１タンパク質をコードする遺伝子及び／又はＧＲＥＭ２タンパク質をコードする遺伝子に特異的にハイブリダイズするオリゴヌクレオチド又はポリヌクレオチドである、（４）に記載のキット。
（６）前記特異的親和性を有する物質が、ＧＲＥＭ１タンパク質及び／又はＧＲＥＭ２タンパク質に対する抗体である、（４）に記載のキット。
（７）前記品質が分化能である（４）〜（６）のいずれかに記載のキット。
（８）(ａ)哺乳動物の幹細胞におけるＧＲＥＭ１タンパク質及び／又はＧＲＥＭ２タンパク質、又は、ＧＲＥＭ１タンパク質をコードする遺伝子及び／又はＧＲＥＭ２タンパク質をコードする遺伝子の発現量を測定する工程と、(ｂ)工程(ａ)で測定したＧＲＥＭ１タンパク質及び／又はＧＲＥＭ２タンパク質、又は、ＧＲＥＭ１タンパク質をコードする遺伝子及び／又はＧＲＥＭ２タンパク質をコードする遺伝子の発現量と基準発現量を比較し、比較結果に基づき品質が高いと評価した幹細胞を採取する工程を含む、品質の高い幹細胞集団の調製方法。
（９）前記幹細胞が間葉系幹細胞である（８）に記載の方法。
（１０）前記品質が分化能である（８）又は（９）に記載の方法。
That is, the present invention includes the following inventions.
(1) A method for evaluating the quality of mammalian stem cells using as an index the expression level of a GREM1 protein and / or GREM2 protein, or a gene encoding a GREM1 protein and / or a gene encoding a GREM2 protein.
(2) The method according to (1), wherein the stem cell is a mesenchymal stem cell.
(3) The method according to (1) or (2), wherein the quality is differentiation ability.
(4) Stem cell quality evaluation kit comprising at least one GREM1 protein and / or GREM2 protein, or a gene encoding the GREM1 protein and / or a substance having specific affinity for the gene encoding the GREM2 protein .
(5) The kit according to (4), wherein the substance having specific affinity is an oligonucleotide or a polynucleotide that specifically hybridizes to a gene encoding the GREM1 protein and / or a gene encoding the GREM2 protein. .
(6) The kit according to (4), wherein the substance having specific affinity is an antibody against GREM1 protein and / or GREM2 protein.
(7) The kit according to any one of (4) to (6), wherein the quality is differentiation ability.
(8) (a) measuring the expression level of GREM1 protein and / or GREM2 protein, or a gene encoding GREM1 protein and / or a gene encoding GREM2 protein in a mammalian stem cell; and (b) step ( The expression level of the GREM1 protein and / or GREM2 protein or the gene encoding the GREM1 protein and / or the gene encoding the GREM2 protein measured in a) is compared with the reference expression level, and the quality is evaluated based on the comparison result. A method for preparing a high-quality stem cell population, comprising the step of collecting the obtained stem cells.
(9) The method according to (8), wherein the stem cell is a mesenchymal stem cell.
(10) The method according to (8) or (9), wherein the quality is differentiation ability.
本発明の方法によれば、幹細胞の分化能を精度よく評価することができる。本発明の方法によって分化能が高いと評価された幹細胞及び当該幹細胞から分化誘導された各種細胞は、再生医療材料、再生美容材料、又は研究応用のための材料として提供することができる。幹細胞を再生医療材料、再生美容材料、又は研究応用のための材料として使用する場合、その分化能などの品質の評価には、インビトロで長期間培養して分化誘導させてその結果を確認する必要があったが、本発明の方法によれば、幹細胞の評価を短時間かつ簡便に効率よく行うことができる。よって、本発明は幹細胞の安全かつ確実な品質管理手段として有用である。 According to the method of the present invention, the differentiation ability of stem cells can be accurately evaluated. Stem cells evaluated as having high differentiation potential by the method of the present invention and various cells induced to differentiate from the stem cells can be provided as regenerative medical materials, regenerative cosmetic materials, or materials for research applications. When stem cells are used as regenerative medical materials, regenerative beauty materials, or materials for research applications, it is necessary to incubate them for a long period of time in vitro to confirm their results in order to evaluate the quality such as differentiation potential However, according to the method of the present invention, stem cells can be evaluated efficiently in a short time and simply. Therefore, the present invention is useful as a safe and reliable quality control means for stem cells.
１．幹細胞の品質を評価する方法
本発明は、ＧＲＥＭ１タンパク質及び／又はＧＲＥＭ２タンパク質、又は、ＧＲＥＭ１タンパク質をコードする遺伝子及び／又はＧＲＥＭ２タンパク質をコードする遺伝子の発現量を指標として幹細胞の品質、特にその分化能を評価する方法を提供する。本発明において評価する幹細胞の「分化能」とは、未分化な幹細胞が機能する細胞へと分化する能力をいう。例えば、脂肪組織に存在する脂肪幹細胞は、脂肪細胞、骨芽細胞、軟骨細胞、平滑筋細胞等（以下、これらの細胞を「分化細胞」という）への分化能を有する。つまり、幹細胞の分化能は、再生医療や研究に用いる上で重要な能力であり、幹細胞の品質に関わる大きな要素となる。
1. TECHNICAL FIELD The present invention relates to the quality of stem cells, particularly the differentiation ability thereof, using the expression level of GREM1 protein and / or GREM2 protein, or a gene encoding GREM1 protein and / or a gene encoding GREM2 protein as an index. Provide a way to evaluate. The “differentiation ability” of a stem cell evaluated in the present invention refers to the ability of an undifferentiated stem cell to differentiate into a functioning cell. For example, adipose stem cells present in adipose tissue have the ability to differentiate into adipocytes, osteoblasts, chondrocytes, smooth muscle cells, etc. (hereinafter these cells are referred to as “differentiated cells”). In other words, the differentiation ability of stem cells is an important ability for use in regenerative medicine and research, and is a major factor related to the quality of stem cells.
本発明に係る幹細胞の品質評価方法を用いて評価する幹細胞としては、本発明の目的に沿うものであれば特に限定されず、例えば胚性の幹細胞（ＥＳ細胞）；骨髄、血液、皮膚（表皮、真皮、皮下組織）、脂肪、毛包、脳、神経、肝臓、膵臓、腎臓、筋肉やその他の組織に存在する体性の幹細胞；遺伝子導入等により人工的に作製された幹細胞（人工多能性幹細胞：ｉＰＳ細胞）が挙げられるが、皮膚又は間葉系組織由来の幹細胞が好ましい。間葉系組織としては、代表的には脂肪組織のほか、筋肉、軟骨、骨等の組織が挙げられる。脂肪組織は、脂肪細胞から成る結合組織で、エネルギー貯蔵のほか、外界からの物理的衝撃や温度変化に対する身体の保護、ホルモンやサイトカインなどを分泌する働きを有する。脂肪組織には幹細胞が存在しており、近年、再生医学への応用も進められている（Ｊａｐａｎｅｓｅ Ｊｏｕｒｎａｌ ｏｆ Ｔｒａｎｓｆｕｓｉｏｎ ａｎｄ Ｃｅｌｌ Ｔｈｅｒａｐｙ， Ｖｏｌ． ５９． Ｎｏ． ３ ５９（３）：４５０―４５６， ２０１３）。 Stem cells evaluated using the stem cell quality evaluation method according to the present invention are not particularly limited as long as they meet the object of the present invention. For example, embryonic stem cells (ES cells); bone marrow, blood, skin (epidermis) , Dermis, subcutaneous tissue), fat, hair follicle, brain, nerve, liver, pancreas, kidney, muscle and other tissues somatic stem cells; stem cells artificially produced by gene transfer (artificial pluripotency) Stem cells: iPS cells), stem cells derived from skin or mesenchymal tissue are preferred. Typical mesenchymal tissues include tissues such as muscle, cartilage and bone in addition to adipose tissue. Adipose tissue is a connective tissue composed of adipocytes, and has functions of storing energy, protecting the body against physical shocks from outside and temperature changes, and secreting hormones and cytokines. Stem cells are present in adipose tissue, and in recent years, application to regenerative medicine has been promoted (Japan Journal of Transfusion and Cell Therapy, Vol. 59. No. 359 (3): 450-456, 2013). .
本発明に係る幹細胞の評価方法の対象となる幹細胞は、幹細胞の分化の方向性及び分化の過程等について同等の特性を持っていれば、その由来は特に限定はされず、例えば、ヒト、サル、マウス、ラット、モルモット、ウサギ、ネコ、イヌ、ウマ、ウシ、ヒツジ、ヤギ、ブタ等の哺乳動物の幹細胞が挙げられる。 The stem cell as a target of the method for evaluating a stem cell according to the present invention is not particularly limited in its origin as long as it has equivalent characteristics with respect to the direction of differentiation of the stem cell and the process of differentiation. And stem cells of mammals such as mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats and pigs.
本発明において幹細胞の品質の評価の指標として用いるＧＲＥＭ１（ｇｒｅｍｌｉｎ １）、ＧＲＥＭ２（ｇｒｅｍｌｉｎ ２）は、Ｄａｎファミリーに属する分泌糖タンパク質で、骨形成タンパク質（ＢＭＰ）であるＢＭＰ２、ＢＭＰ４、ＢＭＰ７の拮抗物質として、ＴＧＦβシグナル伝達経路に作用し、器官形成、組織分化の制御に関与すると考えられている。ヒトのＧＲＥＭ１遺伝子の塩基配列は配列表の配列番号１に示され（Ｇｅｎｂａｎｋ ｎｕｍｂｅｒ：Ｎｕｃｌｅｏｔｉｄｅ ＮＭ＿０１３３７２．６）、ヒトのＧＲＥＭ２遺伝子の塩基配列は配列表の配列番号３にそれぞれ示される（Ｇｅｎｂａｎｋ ｎｕｍｂｅｒ：Ｎｕｃｌｅｏｔｉｄｅ ＮＭ＿０２２４６９．３）。これらの遺伝子は、哺乳動物の種類等によってその塩基配列が異なる場合があり、本発明においては幹細胞の品質の指標として用いることができる限り、配列番号１、３の各塩基配列に対して８０％以上、好ましくは９０％以上、より好ましくは９５％以上の配列同一性を有する塩基配列からなる遺伝子であってもよく、そのようなホモログ遺伝子も、本発明にいうＧＲＥＭ１タンパク質をコードする遺伝子（以下、ＧＲＥＭ１遺伝子という）及びＧＲＥＭ２タンパク質をコードする遺伝子（以下、ＧＲＥＭ２遺伝子という）に包含されるものとする。ＧＲＥＭ１タンパク質のアミノ酸配列は配列表の配列番号２に示され（Ｇｅｎｂａｎｋ ｎｕｍｂｅｒ：Ｐｒｏｔｅｉｎ ＮＰ＿０３７５０４．１）、ＧＲＥＭ２タンパク質のアミノ酸配列は配列表の配列番号４にそれぞれ示される（Ｇｅｎｂａｎｋ ｎｕｍｂｅｒ：Ｐｒｏｔｅｉｎ ＮＰ＿０７１９１４．３）。これらのタンパク質もまた、本発明においては幹細胞の品質の指標として用いることができる限り、配列番号２、４の各アミノ酸配列に対して８０％以上、好ましくは９０％以上、より好ましくは９５％以上の配列同一性を有するアミノ酸配列からなるタンパク質であってもよく、そのようなホモログタンパク質も、本発明にいうＧＲＥＭ１タンパク質及びＧＲＥＭ２タンパク質に包含されるものとする。 In the present invention, GREM1 (gremlin 1) and GREM2 (gremlin 2) used as an index for evaluating the quality of stem cells are secreted glycoproteins belonging to the Dan family and antagonists of BMP2, BMP4 and BMP7 which are bone morphogenetic proteins (BMP) It is thought that it acts on the TGFβ signaling pathway and is involved in the control of organogenesis and tissue differentiation. The base sequence of the human GREM1 gene is shown in SEQ ID NO: 1 (Genbank number: Nucleotide NM — 01372.6), and the base sequence of the human GREM2 gene is shown in SEQ ID NO: 3 of the sequence listing (Genbank number: Nucleotide). NM_022469.3). These genes may have different base sequences depending on the type of mammal, etc., and as long as they can be used as indicators of stem cell quality in the present invention, 80% of each base sequence of SEQ ID NOs: 1 and 3 is used. Above, preferably 90% or more, more preferably 95% or more of a gene comprising a base sequence having sequence identity, such a homologous gene is also a gene encoding the GREM1 protein (hereinafter referred to as the present invention) , GREM1 gene) and a gene encoding a GREM2 protein (hereinafter referred to as GREM2 gene). The amino acid sequence of the GREM1 protein is shown in SEQ ID NO: 2 (Genbank number: Protein NP_037504.1), and the amino acid sequence of the GREM2 protein is shown in SEQ ID NO: 4 of the sequence listing (Genbank number: Protein NP_071914.3). . As long as these proteins can also be used as an indicator of stem cell quality in the present invention, they are 80% or more, preferably 90% or more, more preferably 95% or more with respect to each amino acid sequence of SEQ ID NOs: 2 and 4. It may be a protein consisting of an amino acid sequence having the following sequence identity, and such homologous protein is also included in the GREM1 protein and GREM2 protein referred to in the present invention.
本発明に係る幹細胞の品質の評価方法は、幹細胞におけるＧＲＥＭ１遺伝子及び／又はＧＲＥＭ２遺伝子の発現量が、幹細胞の分化能と負に相関するという知見に基づき、幹細胞の分化能を評価するものである。よって、評価にあたっては、幹細胞におけるＧＲＥＭ１及び／又はＧＲＥＭ２の発現量の測定を行う。ここで、「ＧＲＥＭ１及び／又はＧＲＥＭ２の発現量」とは、ＧＲＥＭ１遺伝子及びＧＲＥＭ２遺伝子の少なくとも一方若しくは両方の発現量、又はＧＲＥＭ１タンパク質及びＧＲＥＭ２タンパク質の少なくとも一方若しくは両方の発現量をいう。幹細胞におけるＧＲＥＭ１及び／又はＧＲＥＭ２の発現量は、絶対値又は相対値（比較対照又は基準発現量との比率や差など）として算出され、必ずしもＧＲＥＭ１及び／又はＧＲＥＭ２の絶対的な発現量を測定する必要はなく、対照のＧＲＥＭ１及び／又はＧＲＥＭ２の発現量との相対的な関係が明らかになればよい。 The method for evaluating the quality of stem cells according to the present invention evaluates the differentiation potential of stem cells based on the knowledge that the expression level of GREM1 gene and / or GREM2 gene in stem cells is negatively correlated with the differentiation potential of stem cells. . Therefore, in the evaluation, the expression level of GREM1 and / or GREM2 in stem cells is measured. Here, “the expression level of GREM1 and / or GREM2” refers to the expression level of at least one or both of the GREM1 gene and the GREM2 gene, or the expression level of at least one or both of the GREM1 protein and the GREM2 protein. The expression level of GREM1 and / or GREM2 in the stem cell is calculated as an absolute value or a relative value (such as a ratio or difference from a comparison control or a reference expression level), and the absolute expression level of GREM1 and / or GREM2 is not necessarily measured It is not necessary, and the relative relationship with the expression level of control GREM1 and / or GREM2 only needs to be clarified.
本発明において、遺伝子の発現量は、当業者に公知の任意の方法により測定することができ、また、測定は、各方法の常法に従って実施すればよい。遺伝子の発現量とは、遺伝子の転写産物であるｍＲＮＡ量をいう。ｍＲＮＡ量の測定は、所望のｍＲＮＡ量を測定できる方法であれば特に限定されず、公知の方法から適宜選択して用いることができる。例えば、ＧＲＥＭ１遺伝子及び／又はＧＲＥＭ２遺伝子にハイブリダイズするオリゴヌクレオチドをプライマーとした遺伝子増幅法、又は、ＧＲＥＭ１遺伝子及び／又はＧＲＥＭ２遺伝子にハイブリダイズするオリゴ（ポリ）ヌクレオチドをプローブとしたハイブリダイゼーション法を利用することができる。具体的には、ＲＴ−ＰＣＲ法、リアルタイムＲＴ−ＰＣＲ法、ＤＮＡマイクロアレイ法、セルアレイ法、組織アレイ法、ノーザンブロット法、ドットブロット法、ＲＮアーゼプロテクションアッセイ法などが挙げられる。上記の測定方法に用いるプライマーやプローブは、標識し、当該標識のシグナル強度を調べることによりｍＲＮＡ量を測定することができる。なかでも、リアルタイムＲＴ−ＰＣＲ法はＲＮＡを直接サンプルに使用でき、遺伝子増幅過程を光学的に測定することで増幅に必要な温度サイクルの回数から遺伝子定量が可能である上で好ましい。また、コントロールとして、ハウスキーピング遺伝子であるＧＡＰＤＨや、ベータアクチンなどのｍＲＮＡの発現量を用い、ＧＲＥＭ１遺伝子及びＧＲＥＭ２遺伝子の発現量を標準化することができる。なお、上記の測定方法に用いるプライマー及びプローブは前記のＧＲＥＭ１遺伝子の塩基配列（配列番号１）及びＧＲＥＭ２遺伝子の塩基配列（配列番号３）、ならびにＧｅｎｂａｎｋなどのデータベースよりそれらのアイソタイプ遺伝子のアクセッション番号により登録された１種以上の塩基配列に基づいて当業者であれば適宜設計し、調製することができる。上記の測定方法は、各方法について様々なプロトコルが報告されており、当業者であれば公知のプロトコルに従い、又は公知のプロトコルを適宜修正や変更を行い実施することができる。 In the present invention, the expression level of a gene can be measured by any method known to those skilled in the art, and the measurement may be performed according to a conventional method of each method. The expression level of a gene refers to the amount of mRNA that is a transcription product of the gene. The measurement of the amount of mRNA is not particularly limited as long as it can measure the desired amount of mRNA, and can be appropriately selected from known methods. For example, gene amplification method using oligonucleotide hybridizing to GREM1 gene and / or GREM2 gene as primer, or hybridization method using oligo (poly) nucleotide hybridizing to GREM1 gene and / or GREM2 gene as probe can do. Specifically, RT-PCR method, real-time RT-PCR method, DNA microarray method, cell array method, tissue array method, Northern blot method, dot blot method, RNase protection assay method and the like can be mentioned. Primers and probes used in the above measurement method can be labeled, and the amount of mRNA can be measured by examining the signal intensity of the label. Among these, the real-time RT-PCR method is preferable because RNA can be directly used for a sample, and gene quantification can be performed from the number of temperature cycles required for amplification by optically measuring the gene amplification process. As controls, the expression levels of GREM1 gene and GREM2 gene can be standardized using expression levels of mRNA such as GAPDH, which is a housekeeping gene, and beta-actin. The primers and probes used in the above measurement method are the base sequence of the GREM1 gene (SEQ ID NO: 1) and the base sequence of the GREM2 gene (SEQ ID NO: 3), and the accession numbers of those isotype genes from databases such as Genbank. Those skilled in the art can design and prepare as appropriate based on one or more kinds of base sequences registered by the above. Various protocols have been reported for the above measurement methods for each method, and those skilled in the art can implement a known protocol according to a known protocol or by appropriately modifying or changing the known protocol.
タンパク質の発現量の測定は、例えば、ＧＲＥＭ１タンパク質及び／又はＧＲＥＭ２タンパク質に対する抗体又は抗体断片を用いて免疫学的に測定する方法を用いることができる。具体的には、ウェスタンブロッティング法、酵素免疫測定法(ＥＬIＳＡ）、放射線免疫測定法（ＲIＡ）、蛍光抗体法、セルアレイ法、組織アレイ法等を挙げることができる。これらの測定方法についても、常法のプロトコール、又は常法のプロトコールを適宜修正・変更したプロトコールによって実施することができる。 The protein expression level can be measured by, for example, an immunological method using an antibody or antibody fragment against the GREM1 protein and / or the GREM2 protein. Specific examples include Western blotting, enzyme immunoassay (ELISA), radioimmunoassay (RIA), fluorescent antibody method, cell array method, tissue array method, and the like. These measurement methods can also be carried out by a conventional protocol or a protocol obtained by appropriately modifying or changing a conventional protocol.
本発明において、幹細胞の品質の評価は、例えば、上記の方法で測定した被験幹細胞におけるＧＲＥＭ１及び／又はＧＲＥＭ２の発現量と、予め定めた基準発現量とを比較することにより行うことができる。基準発現量としては、例えば、一定レベルの分化能を有することが既に確認されている幹細胞（ポジティブコントロール）のＧＲＥＭ１及び／又はＧＲＥＭ２の発現量であってもよく、分化能を有していないことが既に確認されている幹細胞（ネガティブコントロール）の発現量であってもよい。あるいは、幹細胞のＧＲＥＭ１及び／又はＧＲＥＭ２の発現量と分化能との相関図を予め作成しておき、被験幹細胞のＧＲＥＭ１及び／又はＧＲＥＭ２の発現量をその相関図と比較してもよい。発現量の比較は、有意差の有無に基づいて行うことが好ましい。例えば、上記の評価において、被験幹細胞のＧＲＥＭ１及び／又はＧＲＥＭ２の発現量が、基準発現量と比べて、１０％、又は２０％、又は３０％、又は５０％、又は７０％、又は１００％高い、又は低い場合、有意に高い、又は、有意に低いとすることができる。被験幹細胞のＧＲＥＭ１及び／又はＧＲＥＭ２と基準発現量の比較より、被験幹細胞のＧＲＥＭ１及び／又はＧＲＥＭ２の発現量がポジティブコントロールの発現量と同等又はそれより有意に低い場合には、該幹細胞の品質（分化能）が高いと評価でき、被験幹細胞のＧＲＥＭ１及び／又はＧＲＥＭ２の発現量がネガティブコントロールの発現量と同等又はそれより有意に高い場合には、該幹細胞の品質（分化能）が低いと評価できる。また、評価の他の方法としては、ＧＲＥＭ１及び／又はＧＲＥＭ２の発現量のカットオフ値を予め設定しておき、被験幹細胞について測定したＧＲＥＭ１及び／又はＧＲＥＭ２の発現量とカットオフ値とを比較してもよい。カットオフ値は、例えば、ＧＲＥＭ１及び／又はＧＲＥＭ２の発現量と分化能との相関を示す回帰直線（例えば後記実施例の図１）に基づき、所望の分化能を与えるＧＲＥＭ１及び／又はＧＲＥＭ２の発現量とすることができる。例えば、被験幹細胞のＧＲＥＭ１及び／又はＧＲＥＭ２の発現量が前記カットオフ値以下である場合には、該幹細胞の品質（分化能）が高いと評価でき、前記カットオフ値以上である場合には、該幹細胞の品質（分化能）が低いと評価できる。 In the present invention, the quality of stem cells can be evaluated, for example, by comparing the expression level of GREM1 and / or GREM2 in the test stem cells measured by the above method with a predetermined reference expression level. The reference expression level may be, for example, the expression level of GREM1 and / or GREM2 of a stem cell (positive control) that has already been confirmed to have a certain level of differentiation ability, and has no differentiation ability May be the expression level of a stem cell (negative control) that has already been confirmed. Alternatively, a correlation diagram between the expression level of GREM1 and / or GREM2 in stem cells and differentiation potential may be prepared in advance, and the expression level of GREM1 and / or GREM2 in the test stem cell may be compared with the correlation diagram. It is preferable to compare the expression levels based on the presence or absence of a significant difference. For example, in the above evaluation, the expression level of GREM1 and / or GREM2 in the test stem cell is 10%, 20%, 30%, 50%, 70%, or 100% higher than the reference expression level. Or low, it can be significantly higher or significantly lower. From the comparison of the reference expression level with GREM1 and / or GREM2 of the test stem cell, if the expression level of GREM1 and / or GREM2 of the test stem cell is equal to or significantly lower than the expression level of the positive control, the quality of the stem cell ( When the expression level of GREM1 and / or GREM2 of the test stem cell is equal to or significantly higher than the expression level of the negative control, it is evaluated that the quality (differentiation capacity) of the stem cell is low. it can. As another method for evaluation, a cut-off value of the expression level of GREM1 and / or GREM2 is set in advance, and the expression level of GREM1 and / or GREM2 measured for the test stem cell is compared with the cut-off value. May be. The cutoff value is, for example, the expression of GREM1 and / or GREM2 that gives a desired differentiation potential based on a regression line (for example, FIG. 1 in the Examples described later) showing the correlation between the expression level of GREM1 and / or GREM2 and the differentiation potential. It can be an amount. For example, when the expression level of GREM1 and / or GREM2 of the test stem cell is not more than the cut-off value, it can be evaluated that the quality (differentiation ability) of the stem cell is high, and when it is not less than the cut-off value, It can be evaluated that the quality (differentiating ability) of the stem cells is low.
２．幹細胞の品質の評価用キット
本発明に係る幹細胞の品質の評価用キットは、前記の幹細胞のＧＲＥＭ１及び／又はＧＲＥＭ２の発現量を遺伝子レベル又はタンパク質レベルで測定するための試薬を含む。当該キットを構成する試薬としては、ＧＲＥＭ１遺伝子及び／又はＧＲＥＭ２遺伝子、又は、ＧＲＥＭ１タンパク質及び／又はＧＲＥＭ２タンパク質に対して特異的親和性を有する物質、具体的には、ＧＲＥＭ１遺伝子及び／又はＧＲＥＭ２遺伝子に特異的にハイブリダイズするオリゴヌクレオチド又はポリヌクレオチド、ＧＲＥＭ１タンパク質及び／又はＧＲＥＭ２タンパク質に対する抗体を用いることができる。
2. Stem Cell Quality Evaluation Kit The stem cell quality evaluation kit according to the present invention includes a reagent for measuring the expression level of GREM1 and / or GREM2 of the stem cell at the gene level or protein level. Reagents constituting the kit include GREM1 gene and / or GREM2 gene, or a substance having specific affinity for GREM1 protein and / or GREM2 protein, specifically, GREM1 gene and / or GREM2 gene. Antibodies that specifically hybridize to oligonucleotides or polynucleotides, GREM1 protein and / or GREM2 protein can be used.
ＧＲＥＭ１遺伝子及び／又はＧＲＥＭ２遺伝子に特異的にハイブリダイズするオリゴヌクレオチド又はポリヌクレオチドとしては、例えば、配列番号１、３にそれぞれ示される塩基配列又はその相補配列において連続する少なくとも１５塩基以上のオリゴヌクレオチド又はポリヌクレオチドが挙げられ、オリゴヌクレオチドはＧＲＥＭ１遺伝子及び／又はＧＲＥＭ２遺伝子増幅のためのプライマーとして、またポリヌクレオチドはＧＲＥＭ１遺伝子及び／又はＧＲＥＭ２遺伝子の検出のためのプローブとして用いることができる。プライマーの長さは、１５ｂｐ〜５０ｂｐ、好ましくは１５ｂｐ〜３５ｂｐ、より好ましくは２０ｂｐ〜３０ｂｐが例示できる。またプローブの長さは、１５ｂｐ〜全配列の塩基数、好ましくは５０ｂｐ〜１０００ｂｐ、より好ましくは１００ｂｐ〜５００ｂｐが例示できる。オリゴヌクレオチド又はポリヌクレオチドは、測定方法に応じて標識物質をつけてもよい。標識物質としては、当該技術分野においてよく知られる蛍光物質、放射性同位体、化学発光物質、酵素、ビオチン等を用いることができる。 Examples of the oligonucleotide or polynucleotide that specifically hybridizes to the GREM1 gene and / or the GREM2 gene include, for example, oligonucleotides having at least 15 bases that are continuous in the base sequences shown in SEQ ID NOs: 1 and 3 or their complementary sequences, or Polynucleotides can be mentioned, oligonucleotides can be used as primers for GREM1 gene and / or GREM2 gene amplification, and polynucleotides can be used as probes for detection of GREM1 gene and / or GREM2 gene. The length of the primer is 15 bp to 50 bp, preferably 15 bp to 35 bp, more preferably 20 bp to 30 bp. The length of the probe can be 15 bp to the number of bases of the entire sequence, preferably 50 bp to 1000 bp, more preferably 100 bp to 500 bp. Oligonucleotides or polynucleotides may be labeled according to the measurement method. As the labeling substance, fluorescent substances, radioisotopes, chemiluminescent substances, enzymes, biotin and the like well known in the art can be used.
また、ＧＲＥＭ１タンパク質及び／又はＧＲＥＭ２タンパク質に対する抗体は、当該タンパク質と特異的に結合可能なものであれば特に限定されず、ポリクローナル抗体、モノクローナル抗体のいずれであってもよい。また抗体は、上記のタンパク質に特異的に結合し得る限り、断片であってもよい。抗体の断片としては、例えば、Ｆａｂ断片、Ｆ（ａｂ’）２断片、単鎖抗体(ｓｃＦｖ)等が挙げられる。抗体の作成は、上記タンパク質を抗原として、常法により作製することができる。例えば、ポリクローナル抗体は、抗原を感作した哺乳動物（例えば、ウサギ、ラット、マウス等）から血液を採取し、この血液から公知の方法により血清を分離することによって得られる。また、モノクローナル抗体は、抗原を感作した哺乳動物から抗体産生細胞（脾臓細胞、リンパ節細胞等）を取り出して骨髄腫細胞と細胞融合させ、得られたハイブリドーマをクローニングして、その培養物から回収することによって得られる。また、市販品を用いることもできる。タンパク質の定量のために、これらの抗体を適宜標識してもよい。標識物質は、蛍光色素、放射性同位体、酵素等を使用することができる。 Moreover, the antibody with respect to GREM1 protein and / or GREM2 protein will not be specifically limited if it can couple | bond with the said protein specifically, Any of a polyclonal antibody and a monoclonal antibody may be sufficient. The antibody may be a fragment as long as it can specifically bind to the above protein. Examples of antibody fragments include Fab fragments, F (ab ') 2 fragments, single chain antibodies (scFv), and the like. The antibody can be prepared by a conventional method using the protein as an antigen. For example, a polyclonal antibody can be obtained by collecting blood from a mammal (eg, rabbit, rat, mouse, etc.) sensitized with an antigen and separating the serum from this blood by a known method. Monoclonal antibodies can be obtained by removing antibody-producing cells (spleen cells, lymph node cells, etc.) from a mammal sensitized with an antigen, fusing them with myeloma cells, cloning the resulting hybridoma, and It is obtained by collecting. Commercial products can also be used. These antibodies may be appropriately labeled for protein quantification. As the labeling substance, fluorescent dyes, radioisotopes, enzymes and the like can be used.
本発明のキットには、前記の測定方法においてプライマーやプローブとして用いるポリ（オリゴ）ヌクレオチド、又は抗体のいずれかを少なくとも含んでいればよいが、必要に応じて、ＲＮＡ抽出用試薬、ＰＣＲ用緩衝液やＤＮＡポリメラーゼ等のＰＣＲ用試薬、固定化担体、標識物質、標識の検出に用いられる基質化合物、陽性や陰性の標準試料、キットの使用方法を記載した指示書等を含めることもできる。なお、キット中の試薬は溶液でも凍結乾燥物でもよい。 The kit of the present invention may contain at least either a poly (oligo) nucleotide or an antibody used as a primer or probe in the above-described measurement method, but if necessary, an RNA extraction reagent, a PCR buffer PCR reagents such as liquids and DNA polymerases, immobilization carriers, labeling substances, substrate compounds used for label detection, positive and negative standard samples, instructions describing how to use the kit, and the like can also be included. The reagent in the kit may be a solution or a lyophilized product.
３．幹細胞及び分化細胞の調製と利用
本発明はまた、(ａ)哺乳動物の幹細胞におけるＧＲＥＭ１タンパク質及び／又はＧＲＥＭ２タンパク質、又は、ＧＲＥＭ１タンパク質をコードする遺伝子及び／又はＧＲＥＭ２タンパク質をコードする遺伝子の発現量を測定する工程と、(ｂ)工程(ａ)で測定したＧＲＥＭ１タンパク質及び／又はＧＲＥＭ２タンパク質、又は、ＧＲＥＭ１タンパク質をコードする遺伝子及び／又はＧＲＥＭ２タンパク質をコードする遺伝子の発現量と基準発現量を比較し、比較結果に基づき品質が高いと評価した幹細胞を単離する工程を含む、品質の高い幹細胞集団の調製方法を提供する。(ａ)及び（ｂ）の工程は、前記の幹細胞の品質の評価方法に記載の手順に従って行えばよい。
3. Preparation and Utilization of Stem Cell and Differentiated Cell The present invention also provides (a) GREM1 protein and / or GREM2 protein, or a gene encoding GREM1 protein and / or a gene encoding GREM2 protein in mammalian stem cells. Comparing the expression level of the GREM1 protein and / or GREM2 protein or the gene encoding the GREM1 protein and / or the gene encoding the GREM2 protein with the reference expression level measured in the step (b) (a) The present invention provides a method for preparing a high-quality stem cell population, comprising the step of isolating stem cells evaluated as having high quality based on the comparison result. Steps (a) and (b) may be performed according to the procedure described in the method for evaluating the quality of stem cells.
本発明において品質が高いと評価された幹細胞の維持及び目的とする細胞への分化誘導のための培養方法の条件及び操作は、当該技術分野で常套的な条件及び操作に従って行うことができる。 The conditions and operation of the culture method for maintaining the stem cells evaluated as having high quality in the present invention and inducing differentiation into the target cells can be performed according to conditions and operations routine in the art.
幹細胞の分化誘導のための培地には、幹細胞の生存及び増殖に必要な成分（無機塩、炭水化物、ホルモン、必須アミノ酸、非必須アミノ酸、ビタミン、脂肪酸）を含む基本培地、例えば、Ｄｕｌｂｅｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（Ｄ−ＭＥＭ）、Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ（ＭＥＭ）、ＲＰＭＩ１６４０、Ｂａｓａｌ Ｍｅｄｉｕｍ Ｅａｇｌｅ（ＢＭＥ）、Ｄｕｌｂｅｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ：Ｎｕｔｒｉｅｎｔ Ｍｉｘｔｕｒｅ Ｆ−１２（Ｄ−ＭＥＭ／Ｆ−１２）、Ｇｌａｓｇｏｗ Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ（Ｇｌａｓｇｏｗ ＭＥＭ）、ハンクス液（Ｈａｎｋ’ｓ ｂａｌａｎｃｅｄ ｓａｌｔ ｓｏｌｕｔｉｏｎ）に、分化誘導の目的とする細胞に応じた分化誘導又は促進因子を少なくとも１種添加した培地が用いられる。脂肪細胞分化誘導因子としては、例えば、デキサメタゾン（ＤＥＸ）、３−イソブチル−１−メチルキサンチン（ＩＢＭＸ）、インドメタシン（ＩＤＭ）、インスリン（Ｉｎｓ）、トログリタゾン、ビオチン等が挙げられる。骨芽細胞分化誘導因子としては、例えば、デキサメタゾン（ＤＥＸ）、ハイドロコルチゾン、β−グリセロフォスフェート、アスコルビン酸、ＢＭＰ４、ＢＭＰ２等が挙げられる。軟骨細胞分化誘導因子としては、例えば、ＴＧＦ−β３、デキサメタゾン（ＤＥＸ）、アスコルビン酸二リン酸等が挙げられる。また、上記培地には、細胞の増殖速度を増大させるために、必要に応じて、塩基性線維芽細胞増殖因子（ｂＦＧＦ）、上皮細胞増殖因子（ＥＧＦ）等の増殖因子、腫瘍壊死因子（ＴＮＦ）、ビタミン類、インターロイキン類、インスリン、トランスフェリン、ヘパリン、ヘパラン硫酸、コラーゲン、ウシ血清アルブミン（ＢＳＡ）、フィブロネクチン、プロゲステロン、セレナイト、Ｂ２７−サプリメント、Ｎ２−サプリメント、ＩＴＳ−サプリメント等を添加してもよく、また、抗生物質（ペニシリン、ストレプトマイシン等）などを添加してもよい。培地の各成分は、各々適する方法で滅菌して使用する。 A medium for inducing differentiation of stem cells includes a basic medium containing components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids) necessary for stem cell survival and proliferation, such as Dulbecco's Modified. Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Medium Eagle (BME), Dulbecco's Modified Eagle Medium (NME), Nutrient Medium Medium (NME). (Glasgow MEM), Hank's balanced salt solution Medium differentiation inducing or accelerating factor according to the cell of interest was added at least one is used. Examples of the adipocyte differentiation inducing factor include dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), indomethacin (IDM), insulin (Ins), troglitazone, biotin and the like. Examples of the osteoblast differentiation factor include dexamethasone (DEX), hydrocortisone, β-glycerophosphate, ascorbic acid, BMP4, BMP2, and the like. Examples of the chondrocyte differentiation inducing factor include TGF-β3, dexamethasone (DEX), ascorbic acid diphosphate, and the like. In addition, in order to increase the growth rate of the cells, the above-mentioned medium contains growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), tumor necrosis factor (TNF) as necessary. ), Vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, bovine serum albumin (BSA), fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement, etc. In addition, antibiotics (penicillin, streptomycin, etc.) may be added. Each component of the medium is used after being sterilized by a suitable method.
また、上記以外には、１〜２０％の含有率で血清（例えば、１０％ＦＢＳ）が含まれることが好ましい。しかし、血清はロットの違いにより成分が異なり、その効果にバラツキがあるため、ロットチェックを行った後に使用することが好ましい。 In addition to the above, serum (for example, 10% FBS) is preferably contained at a content of 1 to 20%. However, since serum has different components depending on the lot and there are variations in the effects, it is preferable to use the serum after a lot check.
目的とする細胞の分化誘導因子を添加した分化誘導用培地は市販されており、これらの市販品の培地を用いてもよい。例えば、脂肪細胞分化誘導用培地としては、脂肪細胞誘導用培地（ＴＯＹＯＢＯ社製）、脂肪前駆細胞分化培地：Ｐｒｅａｄｉｐｏｃｙｔｅ Ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ Ｍｅｄｉｕｍ（タカラバイオ社製）、脂肪細胞分化培地ＴＡＤＭ（ＴＯＹＯＢＯ社製）等、骨芽細胞分化誘導用培地としては、骨芽細胞誘導用培地（ＴＯＹＯＢＯ社製）、ブレットキット骨芽細胞分化用培地（三光純薬社製）等、軟骨細胞分化誘導用培地としては、軟骨細胞誘導用培地（ＴＯＹＯＢＯ社製）、ブレットキット軟骨細胞分化用培地（三光純薬社製）等が挙げられる。 Differentiation-inducing media to which a target cell differentiation-inducing factor is added are commercially available, and these commercially available media may be used. For example, as the adipocyte differentiation induction medium, adipocyte induction medium (manufactured by TOYOBO), preadipocyte differentiation medium: Preadipocyte Differentiation Medium (manufactured by Takara Bio), adipocyte differentiation medium TADM (manufactured by TOYOBO), etc. As an osteoblast differentiation medium, an osteoblast induction medium (TOYOBO), a bullet kit osteoblast differentiation medium (Sanko Junyaku Co., Ltd.), etc. Examples include an induction medium (manufactured by TOYOBO), a bullet kit chondrocyte differentiation medium (manufactured by Sanko Junyaku Co., Ltd.), and the like.
幹細胞の培養又は幹細胞に分化を促す場合の容器としては、使い捨てのシャーレを使用することが好ましい。なお、培地の交換は１〜３日毎に１回行うことが好ましい。 A disposable petri dish is preferably used as a container for culturing stem cells or promoting differentiation into stem cells. The medium is preferably exchanged once every 1 to 3 days.
幹細胞の増殖及び分化誘導のための培養温度は、細胞の由来により異なるが、例えばヒト由来の場合３０℃〜４０℃が好ましく、３６〜３８℃がより好ましい。また、ＣＯ２ガス濃度は、例えば約１〜１０％が好ましく、約２〜５％がより好ましい。 Although the culture temperature for stem cell proliferation and differentiation induction varies depending on the origin of the cell, for example, in the case of human origin, 30 to 40 ° C is preferable, and 36 to 38 ° C is more preferable. Further, the CO 2 gas concentration is preferably about 1 to 10%, for example, and more preferably about 2 to 5%.
目的とする細胞への分化誘導の確認は、分化細胞の種類に応じて公知の方法にて行うことができる。例えば、脂肪細胞への分化誘導確認は、細胞をオイルレッドＯで染色する方法、細胞内のトリグリセリド量を測定する方法、細胞内のペルオキシゾーム増殖剤活性化受容体−γ（ＰＰＡＲγ）遺伝子の発現量を測定する方法等により行うことができる。骨芽細胞への分化誘導確認は、細胞のカルシウム沈着量を定量する方法、細胞をアルカリフォスファターゼで染色する方法、細胞のアルカリフォスファターゼ活性を測定する方法、細胞内のオステリックス（Ｏｓｔｅｒｉｘ）の発現量を測定する方法等により行うことができる。軟骨細胞への分化誘導確認は、細胞のグリコサミノグリカン量を定量する方法、細胞をアルシアンブルーで染色する方法、細胞内のＣｏｌｌａｇｅｎ ｔｙｐｅ ＩＩの発現量を測定する方法等により行うことができる。 Confirmation of the differentiation induction into the target cell can be performed by a known method according to the type of the differentiated cell. For example, confirmation of induction of differentiation into adipocytes is performed by staining cells with oil red O, measuring the amount of triglycerides in cells, and expressing intracellular peroxisome proliferator-activated receptor-γ (PPARγ) gene. It can be carried out by a method of measuring the amount. Confirmation of differentiation into osteoblasts includes the method of quantifying the amount of calcium deposited in the cell, the method of staining the cell with alkaline phosphatase, the method of measuring the alkaline phosphatase activity of the cell, and the expression level of osterix in the cell It can be performed by a method of measuring Confirmation of differentiation induction into chondrocytes can be performed by a method of quantifying the amount of glycosaminoglycan in the cell, a method of staining the cell with Alcian blue, a method of measuring the expression level of Collagen type II in the cell, etc. .
本発明により品質が確認された幹細胞は、再生医療や基礎研究に用いることができる。また、本発明において幹細胞から分化誘導させた分化細胞は、生体組織の損傷や障害の治療や美容を目的とした移植材料として用いることができる。例えば分化細胞が脂肪細胞である場合は、豊胸、乳がん切除後の乳房再建、目元や頬のシワやハリの低下の改善等、分化細胞が骨芽細胞の場合は骨折の治療、低身長・骨変形・脚長差を改善するための骨延長術等、分化細胞が軟骨細胞は、関節軟骨の退化・変性・変形その他の異常に関連する疾患（例えば、変形性関節症、関節リウマチ、肩関節周囲炎、顎関節症など）の治療への利用が挙げられる。 Stem cells whose quality has been confirmed by the present invention can be used for regenerative medicine and basic research. In the present invention, the differentiated cells induced to differentiate from the stem cells can be used as a transplant material for the purpose of treatment or cosmetic treatment of damage or damage to living tissue. For example, if differentiated cells are adipocytes, breast augmentation, breast reconstruction after resection of breast cancer, improvement of wrinkles and elasticity of eyes and cheeks, etc., if differentiated cells are osteoblasts, treatment of fractures, short stature / Differentiated cells are chondrocytes, such as osteogenesis, rheumatoid arthritis, shoulder joints, etc., such as osteoarthritis, rheumatoid arthritis, shoulder joint, etc. Use for treatment of peripheral inflammation, temporomandibular disorders, etc.).
また、本発明において品質を確認した幹細胞又は幹細胞から分化誘導させた分化細胞を細胞治療に用いる場合には、レシピエントと同種の動物由来の幹細胞を用いることが好ましい。幹細胞や分化細胞の成体への移植方法は、再生させる部位によって異なるが、例えば、脂肪を例にすると、生理食塩水や培養液に幹細胞又は脂肪細胞を１×１０３〜１×１０７個／ｍＬとなるように調整し、腹腔内や皮下などへの注射やカテーテルなどにより局所注入することにより行うことができる。 In the present invention, when stem cells whose quality has been confirmed or differentiated cells induced to differentiate from stem cells are used for cell therapy, it is preferable to use stem cells derived from the same animal species as the recipient. The method of transplanting stem cells or differentiated cells into adults varies depending on the site to be regenerated. For example, when fat is used as an example, 1 × 10 3 to 1 × 10 7 stem cells or adipocytes are contained in physiological saline or culture solution. It can adjust by adjusting to mL, and can carry out by inject | pouring into the abdominal cavity, subcutaneous, etc., or local injection | pouring by a catheter.
以下に本発明を実施例に基づいてさらに詳細に説明するが、本発明はこれら実施例に限定されるものではない。本実施例で用いた幹細胞は、計２５名の被験者（平均年齢72.8歳）から皮膚科外科手術にて摘出された余剰組織内の正常な皮下脂肪組織から分離した。 Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples. Stem cells used in this example were separated from normal subcutaneous adipose tissue in excess tissue removed from a total of 25 subjects (average age 72.8) by dermatological surgery.
（実施例１）皮下脂肪組織からの幹細胞の分離及びＧＲＥＭ１及びＧＲＥＭ２の遺伝子発現量の測定
（１）皮下脂肪組織からの幹細胞の分離
上記被験者から無菌的に摘出された皮下脂肪組織をＰＢＳ（−）で洗浄し、尖刃刀によりペースト状に細切し、０．２％コラゲナーゼ溶液（新田ゼラチン製）を加え、３７℃で６０分間インキュベートすることで細胞外マトリックスを消化した後、穏やかにピペッティングし細胞を分散させた。この細胞分散液を５０ｍＬ容の遠沈管（Ｆａｌｃｏｎ製）にセルストレーナー（Ｆａｌｃｏｎ製）を通しながら移し、残渣を除去した。さらに、細胞分散液を、５分間遠心分離（１３００ｒｐｍ）した。遠心後、上清画分を除去し、Ｔｒｉｓ−ｂｕｆｆｅｒｅｄ ａｍｍｏｎｉｕｍ ｃｈｌｏｒｉｄｅ（ＡＣＴ溶液）を加えて穏やかにピペッティングし細胞を分散させ赤血球を溶血させ、再び５分間遠心分離し、上清画分を除去した。前記遠心分離により下部に沈殿した細胞ペレットをＰＢＳ（−）に懸濁し、よくピペッティングした後、５分間遠心分離した。この洗浄操作を計２回行い、得られた細胞を培養することにより皮下脂肪組織由来幹細胞を得た。
Example 1 Separation of Stem Cells from Subcutaneous Adipose Tissue and Measurement of Gene Expression Levels of GREM1 and GREM2 (1) Separation of Stem Cells from Subcutaneous Adipose Tissue Subcutaneous adipose tissue aseptically removed from the subject was PBS (- ), Chopped into a paste with a sharp blade, add 0.2% collagenase solution (made by Nitta Gelatin), and digest the extracellular matrix by incubating at 37 ° C for 60 minutes, and then gently The cells were dispersed by pipetting. This cell dispersion was transferred to a 50 mL centrifuge tube (Falcon) through a cell strainer (Falcon) to remove the residue. Further, the cell dispersion was centrifuged (1300 rpm) for 5 minutes. After centrifugation, remove the supernatant fraction, add Tris-buffered ammonium chloride (ACT solution) and gently pipet to disperse the cells, hemolyze the red blood cells, and centrifuge again for 5 minutes to remove the supernatant fraction did. The cell pellet precipitated at the bottom by the above centrifugation was suspended in PBS (−), pipetted well, and then centrifuged for 5 minutes. This washing operation was performed twice in total, and the obtained cells were cultured to obtain subcutaneous adipose tissue-derived stem cells.
（２）個人の幹細胞におけるＧＲＥＭ１及びＧＲＥＭ２の遺伝子発現量の測定
上記の工程で分離した幹細胞の一部を６０ｍｍ ｄｉｓｈに１×１０５個播種し、ヒト脂肪由来幹細胞専用培地キット（ロンザジャパン社製）を用いて、３７℃、５％ＣＯ２条件下で培養した。細胞がコンフルエントな状態になったところで、ＲＮＡｉｓｏ ｐｌｕｓ（ＴＡＫＡＲＡ社製）を用いて、総ＲＮＡの抽出を行った。ＧＲＥＭ１及びＧＲＥＭ２のｍＲＮＡ発現量の測定は、細胞から抽出した総ＲＮＡを基にしてリアルタイムＲＴ−ＰＣＲ法により行った。リアルタイムＲＴ−ＰＣＲ法には、ＰｒｉｍｅＳｃｒｉｐｔ ＲＴ ＭａｓｔｅｒＭｉｘ（ＴＡＫＡＲＡ社製）及びＳＹＢＲ Ｓｅｌｅｃｔ Ｍａｓｔｅｒ Ｍｉｘ（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）を用いた。すなわち、５００ｎｇの総ＲＮＡを逆転写反応後、ＰＣＲ反応（９５℃：１５秒間、６０℃：６０秒間、４０ｃｙｃｌｅｓ）を行った。その他の操作は定められた方法に従って実施した。尚、各遺伝子の発現量の測定に使用したプライマーは次の通りである。
(2) Measurement of GREM1 and GREM2 gene expression levels in individual stem cells A portion of the stem cells separated in the above-mentioned steps are seeded in 1 × 10 5 cells in a 60 mm dish, and a human adipose-derived stem cell culture medium kit (manufactured by Lonza Japan) ) At 37 ° C. and 5% CO 2 . When the cells became confluent, total RNA was extracted using RNAiso plus (manufactured by TAKARA). Measurement of mRNA expression levels of GREM1 and GREM2 was performed by real-time RT-PCR method based on total RNA extracted from cells. For the real-time RT-PCR method, PrimeScript RT MasterMix (manufactured by TAKARA) and SYBR Select Master Mix (manufactured by Life Technologies) were used. That is, 500 ng of total RNA was subjected to a reverse transcription reaction, followed by a PCR reaction (95 ° C .: 15 seconds, 60 ° C .: 60 seconds, 40 cycles). Other operations were carried out in accordance with established methods. The primers used for measuring the expression level of each gene are as follows.
ＧＲＥＭ１用のプライマーセット
ＴＣＣＴＴＴＣＡＧＴＣＣＴＧＣＴＣＣＴＴＣＴ（配列番号５）
ＡＧＧＧＣＡＧＴＴＧＡＧＴＧＴＧＡＣＣＡＴ（配列番号６）
ＧＲＥＭ２用のプライマーセット
ＡＡＧＧＣＡＧＡＧＧＧＡＧＡＧＧＧＡＧＡ（配列番号７）
ＣＡＣＣＡＧＧＡＡＣＡＡＧＧＡＣＡＧＧＧＡ（配列番号８）
ＧＡＰＤＨ用のプライマーセット
ＴＧＣＡＣＣＡＣＣＡＡＣＴＧＣＴＴＡＧＣ（配列番号９）
ＴＣＴＴＣＴＧＧＧＴＧＧＣＡＧＴＧＡＴＧ（配列番号１０）
Primer set TCTTTTCAGTCCTGCTCTCTTCT (SEQ ID NO: 5) for GREM1
AGGGCAGTTGAGTGTGCATCAT (SEQ ID NO: 6)
Primer set AAGGCAGAGGGAGAGGGGAGA for GREM2 (SEQ ID NO: 7)
CACCAGGAACAAGGACAGGGA (SEQ ID NO: 8)
Primer set TGCACCACCAACTGCCTTAGC for GAPDH (SEQ ID NO: 9)
TCTTCTGGGTGGCAGTGATG (SEQ ID NO: 10)
上記プライマーを用いて、各被験者から採取された幹細胞におけるＧＲＥＭ１及びＧＲＥＭ２遺伝子発現量を測定した。内部標準には、ＧＡＰＤＨ遺伝子を使用した。また、遺伝子発現量を相対的に定量するために指標とする幹細胞として、不死化処理された骨髄由来間葉系幹細胞（ＵＥ７Ｔ−１３）を用い、この細胞におけるＧＲＥＭ１及びＧＲＥＭ２遺伝子相対発現量を１．０とし、これに対し、各被験者から採取された幹細胞のＧＲＥＭ１及びＧＲＥＭ２遺伝子相対発現量の値を算出した。 Using the above primers, the expression levels of GREM1 and GREM2 genes in stem cells collected from each subject were measured. The GAPDH gene was used as an internal standard. In addition, immortalized bone marrow-derived mesenchymal stem cells (UE7T-13) are used as stem cells as indices for relatively quantifying gene expression levels, and the relative expression levels of GREM1 and GREM2 genes in these cells are 1 The relative expression levels of GREM1 and GREM2 genes of stem cells collected from each subject were calculated.
（実施例２）各種細胞への分化能の比較
実施例１においてＧＲＥＭ１及びＧＲＥＭ２の遺伝子発現量を解析した各被験者の幹細胞を用いて各種細胞（脂肪細胞、骨芽細胞、軟骨細胞）への分化能を比較した。
（１）脂肪細胞への分化能の比較
各被験者の幹細胞とＵＥ７Ｔ−１３（コントロール）を、脂肪細胞誘導用培地（ＴＯＹＯＢＯ社製）にて、３７℃、５％ＣＯ２の条件で１０日間培養し、脂肪細胞への分化誘導を行った。培養液は、３日毎に新鮮な脂肪細胞誘導用培地に交換した。分化誘導後、Ｃｅｌｌ Ｃｏｕｎｔｉｎｇ Ｋｉｔ−８（同仁化学研究所製）により細胞数を測定し、細胞を４％パラホルムアルデヒド溶液により固定した後、オイルレッドＯ染色（脂肪アッセイキット，コスモバイオ社製）により、細胞内の脂肪蓄積量を測定し、細胞あたりの脂肪蓄積量を求めた。尚、コントロールとして用いたＵＥ７Ｔ−１３の脂肪蓄積量を１．０とし、これに対し、各被験者から採取された幹細胞の分化能を算出した。
(Example 2) Comparison of differentiation ability into various cells Differentiation into various cells (adipocytes, osteoblasts, chondrocytes) using stem cells of each subject analyzed for gene expression levels of GREM1 and GREM2 in Example 1 The performance was compared.
(1) Comparison of differentiation ability into adipocytes Stem cells of each subject and UE7T-13 (control) are cultured in adipocyte induction medium (manufactured by TOYOBO) for 10 days under conditions of 37 ° C. and 5% CO 2. Then, differentiation into adipocytes was induced. The culture medium was replaced with a fresh adipocyte induction medium every 3 days. After differentiation induction, the number of cells was measured by Cell Counting Kit-8 (manufactured by Dojindo Laboratories), and the cells were fixed with 4% paraformaldehyde solution, and then oil red O staining (Fat assay kit, manufactured by Cosmo Bio). Then, the amount of accumulated fat in the cells was measured, and the amount of accumulated fat per cell was determined. In addition, the fat accumulation amount of UE7T-13 used as a control was set to 1.0, and the differentiation capacity of stem cells collected from each subject was calculated.
（２）骨芽細胞への分化能の比較
各被験者の幹細胞とＵＥ７Ｔ−１３（コントロール）を、骨芽細胞誘導用培地（ＴＯＹＯＢＯ社製）にて、３７℃、５％ＣＯ２の条件で２１日間培養し、骨芽細胞への分化誘導を行った。培地は３日毎に新鮮な骨芽細胞誘導用培地に交換した。分化誘導後、Ｃｅｌｌ Ｃｏｕｎｔｉｎｇ Ｋｉｔ−８により細胞数を測定し、細胞を４％パラホルムアルデヒド溶液により固定した後、１０％ギ酸溶液にてカルシウムを溶出させ、カルシウムＥ−テストワコー（和光純薬社製）により、カルシウム蓄積量を測定し、細胞当たりのカルシウム蓄積量を求めた。尚、コントロールとして用いたＵＥ７Ｔ−１３のカルシウム蓄積量を１．０とし、これに対し、各被験者から採取された幹細胞の分化能を算出した。
(2) Comparison of differentiating ability into osteoblasts The stem cells of each subject and UE7T-13 (control) were used in an osteoblast induction medium (manufactured by TOYOBO) under conditions of 37 ° C. and 5% CO 2. The cells were cultured for days to induce differentiation into osteoblasts. The medium was replaced with a fresh osteoblast induction medium every 3 days. After differentiation induction, the number of cells was measured with Cell Counting Kit-8, the cells were fixed with 4% paraformaldehyde solution, calcium was eluted with 10% formic acid solution, and calcium E-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) ), The calcium accumulation amount was measured, and the calcium accumulation amount per cell was determined. In addition, the calcium accumulation amount of UE7T-13 used as a control was set to 1.0, and the differentiation capacity of stem cells collected from each subject was calculated.
（３）軟骨細胞への分化能の比較
各被験者の幹細胞とＵＥ７Ｔ−１３（コントロール）を、軟骨細胞誘導用培地（ＴＯＹＯＢＯ社製）にて、３７℃、５％ＣＯ２の条件で１４日間培養し、軟骨細胞への分化誘導を行った。培地は３日毎に新鮮な軟骨細胞誘導用培地に交換した。分化誘導後、プロテインキナーゼにより軟骨細胞を溶解し、ＤＮＡ定量キット（コスモバイオ社製）によりＤＮＡ量を定量するとともにＢｌｙｓｃａｎ Ｇｌｙｃｏｓａｍｉｎｏｇｌｙｃａｎ Ａｓｓａｙ Ｋｉｔ（バイオカラー社製）によりグルコサミノグリカン量を測定し、ＤＮＡ量当たりのグリコサミノグリカン量を求めた。尚、コントロールとして用いたＵＥ７Ｔ−１３のグルコサミノグリカン量を１．０とし、これに対し、各被験者から採取された幹細胞の分化能を算出した。
(3) Comparison of differentiation ability into chondrocytes Stem cells of each subject and UE7T-13 (control) were cultured in a chondrocyte induction medium (manufactured by TOYOBO) for 14 days under conditions of 37 ° C. and 5% CO 2. Then, differentiation into chondrocytes was induced. The medium was replaced with a fresh chondrocyte induction medium every 3 days. After induction of differentiation, chondrocytes are lysed with protein kinase, the amount of DNA is quantified with a DNA quantification kit (manufactured by Cosmo Bio), and the amount of glucosaminoglycan is measured with a Blyscan Glycosaminoglycan Assay Kit (manufactured by Biocolor). The amount of glycosaminoglycan per amount was determined. The amount of glucosaminoglycan of UE7T-13 used as a control was set to 1.0, and the differentiation capacity of stem cells collected from each subject was calculated.
以上の実験により、被験者２５人について、脂肪組織から分離した幹細胞のＧＲＥＭ１及びＧＲＥＭ２の遺伝子発現量と各種細胞への分化能を評価した。評価結果を図１に示す。図１に示した通り、幹細胞の分化能には、個人差が存在し、ＧＲＥＭ１及びＧＲＥＭ２の遺伝子発現量と各種細胞への分化能には、各々、有意に負の相関が認められた。つまり、ＧＲＥＭ１及びＧＲＥＭ２の発現が低いほど幹細胞の分化能は高いことが示された。よって、ＧＲＥＭ１及びＧＲＥＭ２は、幹細胞の分化能、すなわち品質を評価できるマーカー であることが示された。 Through the above experiments, the expression levels of GREM1 and GREM2 genes in stem cells isolated from adipose tissue and the ability to differentiate into various cells were evaluated for 25 subjects. The evaluation results are shown in FIG. As shown in FIG. 1, there are individual differences in stem cell differentiation ability, and a significant negative correlation was observed between the gene expression levels of GREM1 and GREM2 and the differentiation ability into various cells. That is, the lower the expression of GREM1 and GREM2, the higher the differentiation potential of stem cells. Therefore, it was shown that GREM1 and GREM2 are markers that can evaluate the differentiation ability of stem cells, that is, the quality.
本発明の方法によれば、幹細胞の品質、特に分化能を精度よく評価することができる。よって、本発明は、幹細胞を用いた再生医療材料・再生美容材料の製造分野及び基礎研究応用において利用できる。 According to the method of the present invention, it is possible to accurately evaluate the quality of stem cells, particularly the differentiation ability. Therefore, the present invention can be used in the field of manufacturing regenerative medical materials and regenerative beauty materials using stem cells and in basic research applications.
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