JP4958094B2 - Homogeneity identification method of mesenchymal stem cells, homogeneous mesenchymal stem cells obtained by using the method - Google Patents

Homogeneity identification method of mesenchymal stem cells, homogeneous mesenchymal stem cells obtained by using the method Download PDF

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JP4958094B2
JP4958094B2 JP2005276565A JP2005276565A JP4958094B2 JP 4958094 B2 JP4958094 B2 JP 4958094B2 JP 2005276565 A JP2005276565 A JP 2005276565A JP 2005276565 A JP2005276565 A JP 2005276565A JP 4958094 B2 JP4958094 B2 JP 4958094B2
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mesenchymal stem
stem cells
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homogeneity
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幸夫 加藤
真佐美 金輪
晃 五十嵐
真依子 原
紘一郎 辻
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Two Cells Co Ltd
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本発明は、継代培養された間葉系幹細胞の均質性識別方法、その方法を利用して得られる均質間葉系幹細胞に関する。   The present invention relates to a method for identifying homogeneity of subcultured mesenchymal stem cells, and a homogeneous mesenchymal stem cell obtained using the method.

間葉系幹細胞は、軟骨、骨、脂肪へ容易に分化する以外に筋肉、セメント、歯周靭帯、腱、神経、肝臓実質細胞へも分化できる。従って、関節軟骨欠損の修復以外に、骨欠損、人工関節の骨への固定、歯周病、顎骨形成、心筋梗塞、難治性皮膚潰瘍などで、間葉系幹細胞が臨床応用されている。また間葉系幹細胞は樹状細胞の分化と機能に影響するため、臓器移植での拒絶反応の抑制に対しても有効であり、臨床例もすでにある。動物モデルでは、脳梗塞、閉塞性動脈硬化症、腎臓疾患などにも有効である。   Mesenchymal stem cells can be differentiated into muscle, cement, periodontal ligament, tendon, nerve, and liver parenchymal cells as well as easily differentiate into cartilage, bone, and fat. Therefore, in addition to repairing articular cartilage defects, mesenchymal stem cells are clinically applied to bone defects, fixation of artificial joints to bones, periodontal disease, jawbone formation, myocardial infarction, intractable skin ulcers, and the like. In addition, since mesenchymal stem cells affect the differentiation and function of dendritic cells, they are also effective in suppressing rejection in organ transplantation, and there are clinical cases. In animal models, it is also effective for cerebral infarction, obstructive arteriosclerosis, kidney disease and the like.

このように間葉系幹細胞は、非常に多くの治療対象への応用がなされ、また期待もされているが、例えば、骨髄から採取できる間葉系幹細胞の量は非常に少なく採取された骨髄細胞のわずか0.01〜0.001％といわれる。このため、社会的要請に応えるには、まず間葉系幹細胞の継代培養を行って十分な量の間葉系幹細胞を確保することであり、しかもその継代培養は未分化の状態を維持して継続して行うことができ、かつ、使用に際してはその継代培養された細胞が所用の機能を有する細胞に分化し得るものであり、分化能力のない細胞等を含まないような均質な間葉系幹細胞であることが要請される。   As described above, mesenchymal stem cells have been applied to and expected to be applied to a large number of treatment subjects. For example, the amount of mesenchymal stem cells that can be collected from the bone marrow is very small. It is said that it is only 0.01-0.001%. Therefore, in order to meet social demands, it is first necessary to secure a sufficient amount of mesenchymal stem cells by subculturing mesenchymal stem cells, and the subculture maintains an undifferentiated state. Can be continuously performed, and in use, the subcultured cells can be differentiated into cells having a desired function, and do not contain cells that do not have differentiation ability. It is required to be a mesenchymal stem cell.

一方、多くの研究から、骨髄液中の間葉系細胞（培養皿接着細胞）は形態的にかつ機能的に不均質な細胞集団であり、多分化能を持つ間葉系幹細胞以外に、線維芽細胞や各種の前駆細胞（すでに分化が決定されているものの分化状態には至っていない細胞）も含まれていると考えられている。しかし移植用細胞に、これらの間葉系幹細胞以外の細胞が混入すると臨床効果を低下させるとともに、臨床評価の解析をも困難にする。したがって同一性が保証された均質な間葉系幹細胞のみの細胞集団を移植用細胞として用いなければならない。   On the other hand, from many studies, mesenchymal cells (culture dish-adhered cells) in bone marrow fluid are morphologically and functionally heterogeneous cell populations. In addition to multipotent mesenchymal stem cells, fibroblasts And various progenitor cells (cells that have already been determined to differentiate but have not yet reached differentiation). However, when cells other than these mesenchymal stem cells are mixed into the cells for transplantation, the clinical effect is lowered and the analysis of clinical evaluation becomes difficult. Therefore, a cell population of only homogeneous mesenchymal stem cells with guaranteed identity must be used as cells for transplantation.

このような社会的要請に答えるものとして、特許文献１に、骨芽細胞に分化しやすい間葉系幹細胞と、脂肪細胞に分化しやすい間葉系幹細胞とをそれぞれ得る方法が開示されている。すなわち、培養液で完全に満たした培養器中で脂肪細胞集団を培養器の天井面に接触させて培養し、当該培養器の下床面上で増殖した線維芽様の間葉系幹細胞を継代培養することによって骨芽細胞に分化しやすい間葉系幹細胞を得ることができる。また、培養液で完全に満たした培養器中で脂肪細胞集団を培養器の天井面に接触させて培養し、当該培養器の天井面で増殖した線維芽様の間葉系幹細胞を継代培養することによって脂肪細胞に分化しやすい間葉系幹細胞を得ることができることが開示されている。   As a response to such social demands, Patent Document 1 discloses a method for obtaining mesenchymal stem cells that are easily differentiated into osteoblasts and mesenchymal stem cells that are easily differentiated into adipocytes. That is, the adipocyte population is cultured in contact with the ceiling surface of the incubator in an incubator completely filled with the culture solution, and fibroblast-like mesenchymal stem cells grown on the lower floor surface of the incubator are subcultured. By performing subculture, mesenchymal stem cells that are easily differentiated into osteoblasts can be obtained. In addition, the adipocyte population is cultured in contact with the ceiling surface of the incubator in an incubator completely filled with the culture medium, and fibroblast-like mesenchymal stem cells grown on the ceiling surface of the incubator are subcultured. It is disclosed that mesenchymal stem cells that are easily differentiated into adipocytes can be obtained.

特許文献２には、間葉系幹細胞を線維芽細胞増殖因子（FGF）を培地に添加して継代培養を行った細胞について、回数３〜１２の継代を重ねた細胞を分化誘導することにより軟骨分化、骨分化及び脂肪分化のいずれにも分化するとともに、分化誘導しない場合にはいずれの分化もしないことが開示されている。   Patent Document 2 discloses that cells that have been passaged 3 to 12 times are induced to differentiate with respect to cells in which mesenchymal stem cells have been subcultured by adding fibroblast growth factor (FGF) to the medium. Is disclosed to differentiate into any of cartilage differentiation, bone differentiation and fat differentiation and not to undergo differentiation when no differentiation induction is performed.

また、特許文献３には、骨髄液から採取した有核細胞を培養し、ディッシュ底面に付着した接着細胞を剥離したものを初代培養し、これを１〜３回継代培養しそれぞれ軟骨分化をさせた場合に、初代培養のものは全く軟骨分化をせず、１回目継代培養したものは十分に軟骨分化せず、２及び３回目継代培養したものは十分に軟骨分化をすることが開示されている。   In Patent Document 3, nucleated cells collected from the bone marrow fluid are cultured, and the adherent cells attached to the bottom of the dish are peeled off for primary culture, and this is subcultured 1 to 3 times for cartilage differentiation. When cultured, primary cultures do not undergo cartilage differentiation at all, those after the first passage culture do not fully differentiate cartilage, and those after the second and third passage cultures may undergo cartilage differentiation sufficiently. It is disclosed.

特開2004-129549号公報Japanese Patent Laid-Open No. 2004-129549 国際公開第WO2002/22788号International Publication No.WO2002 / 22788 特開2003-289855号公報JP 2003-289855 JP

このような従来技術によれば、1〜3回以上継代培養された間葉系幹細胞の細胞集団は適当な培養方法を選択することにより目的とする分化能を有する間葉系幹細胞を得ることができ、あるいは、脂肪分化、骨分化又は軟骨分化のいずれの分化能をも有する間葉系幹細胞を得ることができることが推測される。   According to such a conventional technique, the mesenchymal stem cell population subcultured 1 to 3 times or more to obtain a mesenchymal stem cell having the desired differentiation potential by selecting an appropriate culture method It is presumed that mesenchymal stem cells having any differentiation ability of adipose differentiation, bone differentiation or cartilage differentiation can be obtained.

しかしながら、このような方法で継代培養された間葉系幹細胞は、脂肪分化能を有するのか、骨分化能を有するのか、軟骨分化能を有するのか、あるいはいずれか２つ又は３つの方向に分化する分化能を有するのかが明確でなく、実際に分化誘導を行って確認しなければならないという問題がある。このため、望ましくは３つの分化能を有し分化誘導しない限りいずれの分化もしない間葉系幹細胞が得られることであるが、そのような性質を有する間葉系幹細胞を予め取得することが困難であるという問題がある。また、従来方法により得られる細胞集団の均質性は不明である。   However, mesenchymal stem cells subcultured by such a method have a fat differentiation ability, a bone differentiation ability, a cartilage differentiation ability, or differentiation in any two or three directions. There is a problem that it is not clear whether it has differentiation ability, and it is necessary to confirm by actually performing differentiation induction. For this reason, it is desirable to obtain mesenchymal stem cells that have three differentiation potentials and do not undergo any differentiation unless differentiation is induced, but it is difficult to obtain mesenchymal stem cells having such properties in advance. There is a problem that. In addition, the homogeneity of the cell population obtained by the conventional method is unknown.

本発明は、係る従来技術の問題点に鑑み、継代培養された間葉系幹細胞が脂肪分化、骨分化及び軟骨分化の３つの方向に分化する分化能を有し、分化誘導しない限りいずれの分化もしない細胞（以下多機能間葉系幹細胞という）であるか否かを識別することができる間葉系幹細胞の均質性識別方法を提供することを目的とする。また、その方法を利用して得られる均質間葉系幹細胞を提供することを目的とする。   In view of the problems of the related art, the present invention has a differentiation ability in which mesenchymal stem cells subcultured are differentiated in three directions of adipose differentiation, bone differentiation, and cartilage differentiation. It is an object of the present invention to provide a method for identifying the homogeneity of mesenchymal stem cells that can identify whether the cells do not differentiate (hereinafter referred to as multifunctional mesenchymal stem cells). Moreover, it aims at providing the homogeneous mesenchymal stem cell obtained using the method.

本発明者等は、骨髄液を培養皿中で培養したときに培養皿に接着し増殖した培養皿接着細胞は、一般にはその培養中に行われた１〜３回の培地交換で造血系細胞は除かれているが、多機能間葉系幹細胞以外に、分化能力のない線維芽細胞様の細胞（CFU-Fの一部）、骨／軟骨／脂肪への一方向だけの分化能をもつ前駆細胞、造血支持能のある間質細胞など、多様な細胞が存在する可能性があるという事実に注目した。そして、使用される間葉系幹細胞がどのような異質の細胞を含むか、あるいはどの程度の均質性を有するかについて判別するには遺伝子分析が有力であり、特に多機能間葉系幹細胞と形態においてほとんど識別できず、しかも培養細胞中に混入のおそれがある線維芽細胞様の細胞（以下線維芽細胞という）の遺伝子発現レベル基準とし、それより高い発現レベルを示す遺伝子の遺伝子発現状況からその細胞集団の識別又は均質性を判別することができるという知見に基づいて本発明を完成した。   The inventors of the present invention have found that culture dish-adherent cells that have grown and adhered to a culture dish when bone marrow fluid is cultured in a culture dish are generally hematopoietic cells obtained by exchanging the medium one to three times during the culture. In addition to multifunctional mesenchymal stem cells, fibroblast-like cells with no differentiation ability (part of CFU-F), having differentiation ability in only one direction to bone / cartilage / fat We focused on the fact that a variety of cells may exist, such as progenitor cells and stromal cells capable of supporting hematopoiesis. In addition, genetic analysis is effective in determining what kind of heterogeneous cells the mesenchymal stem cells are used, and how homogeneous they are, especially with multifunctional mesenchymal stem cells and morphology The fibroblast-like cells (hereinafter referred to as fibroblasts) that can hardly be identified in culture cells and may be mixed in the cultured cells, and the gene expression status of genes that show higher expression levels The present invention has been completed based on the knowledge that cell population discrimination or homogeneity can be determined.

本発明に係る間葉系幹細胞の均質性識別方法は、継代培養された間葉系幹細胞の細胞集団から複数のクローンを作製する段階と、該クローン及びその母集団について所定の遺伝子群に関するmRNA発現強度を取得する段階と、該取得された前記クローン及びその母集団のmRNA発現強度群を前記遺伝子群に対して図式化した遺伝子発現パターンを求める段階と、該得られた遺伝子発現パターンから前記母集団の均質性を判別する段階と、からなる。   The method for identifying homogeneity of mesenchymal stem cells according to the present invention comprises a step of preparing a plurality of clones from a cell population of subcultured mesenchymal stem cells, and mRNA relating to a predetermined gene group for the clone and its population Obtaining an expression intensity, obtaining a gene expression pattern obtained by schematizing the obtained clone and the mRNA expression intensity group of the population thereof with respect to the gene group, and obtaining the gene expression pattern from the obtained gene expression pattern And determining the homogeneity of the population.

また、本発明に係る間葉系幹細胞の均質性識別方法は、継代培養された間葉系幹細胞の細胞集団の所定の遺伝子群に関するmRNA発現強度を取得する段階と、該取得されたmRNA発現強度群を前記遺伝子群に対して図式化した遺伝子発現パターンを求める段階と、該遺伝子発現パターンから前記細胞集団の均質性を判別する段階と、からなる。   Further, the method for identifying the homogeneity of mesenchymal stem cells according to the present invention includes the step of obtaining the mRNA expression intensity for a predetermined gene group of the cell population of the subcultured mesenchymal stem cells, and the obtained mRNA expression It comprises a step of obtaining a gene expression pattern in which an intensity group is diagrammatically represented with respect to the gene group, and a step of determining the homogeneity of the cell population from the gene expression pattern.

このような間葉系幹細胞の均質性識別方法を使用することによって均質な多機能間葉系幹細胞を得ることができる。   By using such a method for identifying mesenchymal stem cell homogeneity, a homogeneous multifunctional mesenchymal stem cell can be obtained.

本発明に係る均質間葉系幹細胞は、脂肪分化、骨分化及び軟骨分化の３つの方向に分化する分化能を有し、分化誘導しない限りいずれの分化もしない基準間葉系幹細胞から得られる所定の遺伝子群に関する遺伝子発現パターンに類似する遺伝子発現パターンを有するものである。   The homogeneous mesenchymal stem cell according to the present invention has a differentiation ability to differentiate in three directions of adipose differentiation, bone differentiation and cartilage differentiation, and is obtained from a predetermined mesenchymal stem cell that does not undergo any differentiation unless differentiation is induced. It has a gene expression pattern similar to the gene expression pattern for these gene groups.

また、本発明に係る均質間葉系幹細胞は、所定の遺伝子群に関する各mRNA発現強度と、該遺伝子群に関し基準間葉系幹細胞から得られる各mRNA発現強度との分散和をとった場合に、その分散和が所定の範囲内にあるものである。   Further, the homogeneous mesenchymal stem cells according to the present invention, when taking the variance sum of each mRNA expression intensity related to the predetermined gene group and each mRNA expression intensity obtained from the reference mesenchymal stem cell related to the gene group, The dispersion sum is within a predetermined range.

上記発明において、基準間葉系幹細胞は、クローン化された細胞であるのがよい。また、所定の遺伝子群は、CTGF、IGFBP7、KCTD12、LAMA3、LIF、MGP、PRG1、TRIB2、IGF1、BMP4、SERPINI1、CD74、HLA-DRA、HLA-DRB、TGM2、DNCI1、MCAM、TFPI2、ARHGDIBのいずれか複数の遺伝子からなる遺伝子群であるのがよい。   In the above invention, the reference mesenchymal stem cell may be a cloned cell. In addition, the predetermined gene group is CTGF, IGFBP7, KCTD12, LAMA3, LIF, MGP, PRG1, TRIB2, IGF1, BMP4, SERPINI1, CD74, HLA-DRA, HLA-DRB, TGM2, DNCI1, MCAM, TFPI2, ARHGDIB A gene group composed of any one of a plurality of genes is preferable.

さらに、所定の遺伝子群として、ADAMTS1、ABHD2、ADD3、ANXA10、ACLY、ATP6V1G3、BDNF、BRIP1、CDH6、CPA4、CDC25A、CHI3L1、F2R、F2RL1、CCND1、CKAP2、DNAH3、SMURF2、EFEMP1、DCBLD、ESM1、EDG2、LOC221810、GABRB1、GATA6、GMFG、HGF、HMGA2、PHLDB2、HTR7、MGC14161、DGKG、PRDM16、MCTP2、FLJ23033、FLJ35681、FLJ38725、C9orf72、LYPDC1、IF、IGFBP1、IGFBP3、IGFBP5、ITGA5、ITGB3、IFI30、IL6、KRT19、KRT23、KRTAP1-5、KRTHA4、KCTD16、KIAA1913、LXN、LEPR、Lrp2bp、MET、MICA、LF1、PR3、TN4、ASK、LAU、CTD4、4HA2、TGER1、ROS1、AB27B、AC2、GS4、ARRES1、LEKHK1、YPN、LC16A4、LC2A1、LC20A1、AMD3、UHW2、YT1、RPC4、CHL1のいずれか複数の遺伝子からなる遺伝子群を選択することができる。   Furthermore, as a predetermined gene group, ADAMTS1, ABHD2, ADD3, ANXA10, ACLY, ATP6V1G3, BDNF, BRIP1, CDH6, CPA4, CDC25A, CHI3L1, F2R, F2RL1, CCND1, CKAP2, DNAH3, SMURF2, EFEMP1, DCBLD, ES EDG2, LOC221810, GABRB1, GATA6, GMFG, HGF, HMGA2, PHLDB2, HTR7, MGC14161, DGKG, PRDM16, MCTP2, FLJ23033, FLJ35681, FLJ38725, C9orf72, LYPDC1, IF, IGFBP1, IGFBP3, IGFBPIT3, IGFBP5, GA5 IL6, KRT19, KRT23, KRTAP1-5, KRTHA4, KCTD16, KIAA1913, LXN, LEPR, Lrp2bp, MET, MICA, LF1, PR3, TN4, ASK, LAU, CTD4, 4HA2, TGER1, ROS1, AB27B, AC2, GS4, A gene group consisting of a plurality of genes of ARRES1, LEKHK1, YPN, LC16A4, LC2A1, LC20A1, AMD3, UHW2, YT1, RPC4, and CHL1 can be selected.

本発明に係る間葉系幹細胞の均質性識別方法は、継代培養された間葉系幹細胞を分化誘導しないで予めその細胞集団が多機能間葉系幹細胞であるか否かを識別することができる。そして、この方法を利用することにより均質な特性を有する多機能間葉系幹細胞特性を得ることができる。   The method for identifying homogeneity of mesenchymal stem cells according to the present invention may identify whether or not the cell population is a multifunctional mesenchymal stem cell in advance without inducing differentiation of the subcultured mesenchymal stem cells. it can. By using this method, it is possible to obtain multifunctional mesenchymal stem cell characteristics having homogeneous characteristics.

以下本発明に係る間葉系幹細胞の均質性識別方法の実施形態について説明する。本発明に係る間葉系幹細胞の均質性識別方法は、継代培養された間葉系幹細胞の細胞集団から複数のクローンを作製する段階と、該クローン及びその母集団について所定の遺伝子群に関するmRNA発現強度を取得する段階と、該取得された前記クローン及びその母集団のmRNA発現強度群を前記遺伝子群に対して図式化した遺伝子発現パターンを求める段階と、該得られた遺伝子発現パターンから前記母集団の均質性を判別する段階と、からなる。   Embodiments of the mesenchymal stem cell homogeneity identification method according to the present invention will be described below. The method for identifying homogeneity of mesenchymal stem cells according to the present invention comprises a step of preparing a plurality of clones from a cell population of subcultured mesenchymal stem cells, and mRNA relating to a predetermined gene group for the clone and its population Obtaining an expression intensity, obtaining a gene expression pattern obtained by schematizing the obtained clone and the mRNA expression intensity group of the population thereof with respect to the gene group, and obtaining the gene expression pattern from the obtained gene expression pattern And determining the homogeneity of the population.

本発明において、間葉系幹細胞は、大腿骨、脛骨、腸骨等いずれに由来するものであってもよい。間葉系幹細胞とは、それらのいずれかから採取し培養を行ったとき、培養皿に接着する性質を有し、線維芽細胞様の形態を有する細胞をいう。このようにして得られた間葉系幹細胞は継代培養される。なお、継代培養とは、取得した細胞を所定の稠密状態になるまで培養し、その後その培養環境から一旦取り出し、他の培養環境に移し換えて培養することをいう。初代培養から１回目の継代培養 、１回目から２回目移行の継代培養はそれぞれ同一の培養条件としてもよく、異なる培養条件としてもよい。すなわち、培地組成、培養温度等が異なる培養条件で培養するものであってもよい。   In the present invention, the mesenchymal stem cells may be derived from any of the femur, tibia, iliac and the like. A mesenchymal stem cell refers to a cell that has a property of adhering to a culture dish when collected from any of them and cultured, and has a fibroblast-like morphology. The mesenchymal stem cells thus obtained are subcultured. The subculture means that the obtained cells are cultured until reaching a predetermined dense state, and then taken out from the culture environment, transferred to another culture environment, and cultured. The first subculture from the first culture and the subculture from the first transfer to the second subculture may be the same or different culture conditions. That is, you may culture | cultivate on culture conditions from which a culture medium composition, culture | cultivation temperature, etc. differ.

所定の遺伝子群とは、間葉系幹細胞が有する遺伝子のうち所定のものをいう。この所定の遺伝子は、線維芽細胞よりも高い遺伝子発現を示す遺伝子を選択するのがよい。すなわち、線維芽細胞は、繊維芽様の形態を有し、上記の間葉系幹細胞とは形態的にはほとんど区別ができず、脂肪分化能、骨分化能又は軟骨分化能のいずれの機能をも有しない。しかも、間葉系幹細胞の初代培養時又は継代培養時に混入のおそれがある細胞である。従って、まず、線維芽細胞は間葉系幹細胞集団から排除する必要があり、均質な多機能間葉系幹細胞であるというには、線維芽細胞を含まない細胞集団であることが証明されたものでなければならない。   The predetermined gene group refers to a predetermined gene among genes possessed by mesenchymal stem cells. As this predetermined gene, a gene showing higher gene expression than that of fibroblasts is preferably selected. That is, fibroblasts have a fibroblast-like morphology and are hardly distinguishable morphologically from the above mesenchymal stem cells, and have any function of adipose differentiation ability, bone differentiation ability, or cartilage differentiation ability. Does not have. And it is a cell which may be mixed at the time of the primary culture of a mesenchymal stem cell, or a subculture. Therefore, fibroblasts must first be excluded from the mesenchymal stem cell population, and to be a homogeneous multifunctional mesenchymal stem cell is a cell population that does not contain fibroblasts. Must.

このため、本発明においては、線維芽細胞よりも高い遺伝子発現を示す、例えば、以下に説明する遺伝子略名でCTGF、IGFBP7、KCTD12、LAMA3、LIF、MGP、PRG1、TRIB2、IGF1、BMP4、SERPINI1、CD74、HLA-DRA、HLA-DRB、TGM2、DNCI1、MCAM、TFPI2、ARHGDIBの遺伝子（図２）が選ばれる。対象とされる遺伝子は必ずしもこれらのすべてを含めることを要しない。以下に説明する遺伝子発現パターンが認識できる程度の数の遺伝子が対象にされればよく、少なくとも複数の遺伝子が対象とされる。なお、遺伝子略名TRIB2は、TRB2と表記される場合もある。   Therefore, in the present invention, the gene expression is higher than that of fibroblasts.For example, CTGF, IGFBP7, KCTD12, LAMA3, LIF, MGP, PRG1, TRIB2, IGF1, BMP4, SERPINI1 , CD74, HLA-DRA, HLA-DRB, TGM2, DNCI1, MCAM, TFPI2, and ARHGDIB genes (FIG. 2) are selected. The gene of interest need not necessarily include all of these. The number of genes that can recognize the gene expression pattern described below may be targeted, and at least a plurality of genes are targeted. In addition, the gene abbreviation TRIB2 may be written as TRB2.

上記に示す遺伝子は、一般に高い遺伝子発現強度を示すので有用であるが、以下の遺伝子を対象にすることもできる。すなわち、ADAMTS1、ABHD2、ADD3、ANXA10、ACLY、ATP6V1G3、BDNF、BRIP1、CDH6、CPA4、CDC25A、CHI3L1、F2R、F2RL1、CCND1、CKAP2、DNAH3、SMURF2、EFEMP1、DCBLD、ESM1、EDG2、LOC221810、GABRB1、GATA6、GMFG、HGF、HMGA2、PHLDB2、HTR7、MGC14161、DGKG、PRDM16、MCTP2、FLJ23033、FLJ35681、FLJ38725、C9orf72、LYPDC1、IF、IGFBP1、IGFBP3、IGFBP5、ITGA5、ITGB3、IFI30、IL6、KRT19、KRT23、KRTAP1-5、KRTHA4、KCTD16、KIAA1913、LXN、LEPR、Lrp2bp、MET、MICA、LF1、PR3、TN4、ASK、LAU、CTD4、4HA2、TGER1、ROS1、AB27B、AC2、GS4、ARRES1、LEKHK1、YPN、LC16A4、LC2A1、LC20A1、AMD3、UHW2、YT1、RPC4、CHL1の遺伝子（図３、４）のうちいずれか複数の遺伝子を選択することもできる。   The genes shown above are useful because they generally exhibit high gene expression intensity, but the following genes can also be targeted. ADAMTS1, ABHD2, ADD3, ANXA10, ACLY, ATP6V1G3, BDNF, BRIP1, CDH6, CPA4, CDC25A, CHI3L1, F2R, F2RL1, CCND1, CKAP2, DNAH3, SMURF2, EFEMP1, DCBLD, ESMLOC1, EDG1, B GATA6, GMFG, HGF, HMGA2, PHLDB2, HTR7, MGC14161, DGKG, PRDM16, MCTP2, FLJ23033, FLJ35681, FLJ38725, C9orf72, LYPDC1, IF, IGFBP1, IGFBP3, IGFBP5, ITGA5, ITGB3, IFI30, ILK, RT1, 23 KRTAP1-5, KRTHA4, KCTD16, KIAA1913, LXN, LEPR, Lrp2bp, MET, MICA, LF1, PR3, TN4, ASK, LAU, CTD4, 4HA2, TGER1, ROS1, AB27B, AC2, GS4, ARRES1, LEKHK1, YPN, Any of the genes of LC16A4, LC2A1, LC20A1, AMD3, UHW2, YT1, RPC4, and CHL1 (FIGS. 3 and 4) can be selected.

なお、識別をしようとする間葉系幹細胞と線維芽細胞であることが明確な細胞集団について同時に遺伝子発現を調べることによってその間葉系幹細胞が線維芽細胞を含まない細胞集団であることを証明することができる。また、上記のように線維芽細胞よりも高い遺伝子発現を示す遺伝子についての遺伝子発現に限定することは、初代培養の間葉系幹細胞のようにmRNA発現強度を測定することが不可能なほどに均質性を有しない細胞を除外できるという意義を有する。   It is proved that the mesenchymal stem cell is a cell population that does not contain fibroblasts by simultaneously examining gene expression for a cell population clearly identified as mesenchymal stem cells and fibroblasts to be identified. be able to. In addition, as described above, limiting to gene expression for genes that exhibit higher gene expression than fibroblasts is such that it is impossible to measure mRNA expression intensity as in primary cultured mesenchymal stem cells. It has the significance that cells that do not have homogeneity can be excluded.

遺伝子群に関するmRNA発現強度とは、公知のPCR法、サザンブロッティング法あるいは、DNAマイクロアレイ法等によって求められる遺伝子発現状態の相対的な大きさの程度をいう。遺伝子群に関するmRNA発現強度は、上記のいずれの方法により求められるものであってもよい。   The mRNA expression intensity related to a gene group refers to the degree of relative magnitude of a gene expression state determined by a known PCR method, Southern blotting method, DNA microarray method or the like. The mRNA expression intensity relating to the gene group may be determined by any of the above methods.

図式化とは、遺伝子群によって発現されたmRNA発現強度について全体として視覚的にみればどのような形態（遺伝子発現パターン）を有するか判断するための基礎になるグラフ又は図面をいう。遺伝子の類似度は系統や個体間の類似度を反映しているといわれるように、本発明は、間葉系幹細胞の遺伝子が類似であれば均質であるということに基礎をおいている。すなわち、複数の遺伝子から形成される図式化された遺伝子発現パターンが類似であれば間葉系幹細胞は均質であると判断される。   Schematization refers to a graph or drawing that is the basis for determining what form (gene expression pattern) the mRNA expression intensity expressed by a gene group has as a whole visually. As it is said that the gene similarity reflects the similarity between strains and individuals, the present invention is based on the fact that the genes of the mesenchymal stem cells are homogeneous if they are similar. That is, if the diagrammatic gene expression pattern formed from a plurality of genes is similar, the mesenchymal stem cells are judged to be homogeneous.

この類似性を判断するには基準の間葉系幹細胞との比較により判別するのがよい。そして、その基準となる基準間葉系幹細胞は、クローンであるのが好ましい。すなわち、多機能間葉系幹細胞であることが明らかな基準となるクローンから求めた遺伝子群についてのmRNA発現強度に関する遺伝子パターンと類似な遺伝子パターンを示す間葉系幹細胞はそのクローンと同質であると判断されるからである。なお、クローンとは、一個の細胞から無性生殖的に増殖した細胞をいう。   In order to judge this similarity, it is better to discriminate by comparison with a reference mesenchymal stem cell. The reference mesenchymal stem cell serving as the reference is preferably a clone. That is, a mesenchymal stem cell that shows a similar gene pattern to the mRNA expression intensity of a gene group obtained from a clone that is a clear reference that is a multifunctional mesenchymal stem cell is the same quality as that clone It is because it is judged. A clone refers to a cell that has grown asexually from a single cell.

以下に、本発明に係る間葉系幹細胞の均質性識別方法の一実施例について説明する。図１は、上記の方法により得られた遺伝子発現パターンの一例を示す。図１は、各遺伝子を横軸に表し、縦軸にそのmRNA発現強度を表したグラフである。横軸の遺伝子番号は、図２に示す遺伝子に対応している。例えば、遺伝子番号２の遺伝子は、遺伝子略名がIGFBP7であり、遺伝子名称がinsulin-like growth factor binding protein 7を示す。図中のパラメータにおいて、C1〜C8はクローン番号を示し、massは前記クローンがクローン化される母胎となった母集団を示す。F1〜F4は、上記間葉系幹細胞を採取した人を含め異なる4人から採取したそれぞれの線維芽細胞を示す。なお、クローンC1〜C8は継代3回の細胞集団からクローン化した細胞、母集団はさらに継代した継代4回の細胞集団である。線維芽細胞は、継代回数が6から11の細胞である。 Hereinafter, an embodiment of the method for identifying homogeneity of mesenchymal stem cells according to the present invention will be described. FIG. 1 shows an example of a gene expression pattern obtained by the above method. FIG. 1 is a graph showing each gene on the horizontal axis and the mRNA expression intensity on the vertical axis. The gene numbers on the horizontal axis correspond to the genes shown in FIG. For example, the gene number 2 has the gene abbreviation IGFBP7 and the gene name insulin-like growth factor binding protein 7. In the parameters in the figure, C 1 to C 8 indicate clone numbers, and mass indicates the population that became the mother of the clone. F 1 to F 4 indicate the respective fibroblasts collected from four different people including those who collected the mesenchymal stem cells. In addition, clones C 1 to C 8 are cells cloned from the 3rd passage cell population, and the population is a further 4th passage cell population. Fibroblasts are cells with passage numbers between 6 and 11.

図１によると、クローンC1〜C8の遺伝子のmRNA発現強度とF1〜F4のmRNA発現強度とは強度レベルが異なり、それぞれ明確に区別されていることが分かる。また、クローンC1〜C8は個々の遺伝子をみると遺伝子の種類によってはmRNA発現強度のばらつきはあるが、全体としてみればそれぞれのクローンのmRNA発現強度を折れ線で示す図形（遺伝子パターン）は類似していることが分かる。また、母集団の遺伝子発現パターンをみると、クローンC1〜C8の遺伝子発現パターンに類似していることが分かる。 According to FIG. 1, it can be seen that the mRNA expression intensity of the genes of clones C 1 to C 8 and the mRNA expression intensity of F 1 to F 4 are different in intensity level and are clearly distinguished from each other. Although clone C 1 -C 8 is the variation in the mRNA expression level depending on the type of gene Looking at individual genes, figures showing the mRNA expression level of each clone Come to a whole in a line (gene pattern) You can see that they are similar. In terms of gene expression patterns of the population, it is understood that similar to the gene expression pattern of the clone C 1 -C 8.

このような遺伝子発現パターンの類似性は、他の図形化によっても確認することができる。図５は、図１のクローンC1〜C8の遺伝子のmRNA発現強度について最も高い強度を示す直線を連結した上限線と、最も低い強度を示す直線を連結した下限線とで囲まれる範囲を斜線部で表し、母集団の遺伝子発現パターンを太い破線で示したグラフである。細い破線は、母集団の発現パターンを示す太い破線を上方に平行移動した折れ線である。図３によると、細い破線部のほとんどが斜線部分に含まれ、しかも、その細い破線部は、上限線と下限線のいずれかによく一致することが分かる。すなわち、クローンC1〜C8の遺伝子発現パターンと母集団の遺伝子発現パターンは近似しているということができる。 Such similarity of gene expression patterns can be confirmed by other graphic representations. FIG. 5 shows the range surrounded by the upper limit line connecting the straight lines showing the highest intensity and the lower limit line connecting the straight lines showing the lowest intensity with respect to the mRNA expression intensity of the genes of clones C 1 to C 8 in FIG. It is the graph which showed the gene expression pattern of the population with the thick broken line with the oblique line part. The thin broken line is a broken line obtained by translating upward the thick broken line indicating the expression pattern of the population. According to FIG. 3, it can be seen that most of the thin broken line portion is included in the hatched portion, and that the thin broken line portion coincides well with either the upper limit line or the lower limit line. That is, it can be said that the gene expression patterns of clones C 1 to C 8 are similar to the gene expression pattern of the population.

図６〜図９は、上記の遺伝子発現パターンを示すクローンの母集団（図１、５の太い破線で表される遺伝子発現を示す細胞集団）及び上記の継代培養で得た他の細胞集団について分化能を調べた試験結果を示す。これらの図から各細胞集団は多機能間葉系幹細胞であることが分かり、細胞集団は、脂肪分化、骨分化及び軟骨分化の３つの方向に分化する分化能を有し、分化誘導しない限りいずれの分化もしない細胞であることが分かる。以下に個々の試験結果について具体的に説明する。図６は、骨分化を行った結果、図７及び８は軟骨分化を行った結果、図９は脂肪分化を行った結果を示す。各図において横軸に示す記号は、細胞集団の番号を示し、パラメータの＋は分化誘導を行った細胞集団を示し、−は分化誘導を行わなかった細胞集団を示す。なお、M1〜M8は数人から得た間葉系幹細胞（母集団）の細胞集団記号を示す。 FIGS. 6 to 9 show populations of clones showing the above gene expression patterns (cell populations showing gene expression represented by thick broken lines in FIGS. 1 and 5) and other cell populations obtained by the above subculture. The test result which investigated differentiation ability about is shown. From these figures, it can be seen that each cell population is a multifunctional mesenchymal stem cell, and the cell population has differentiation ability to differentiate in three directions of adipose differentiation, bone differentiation and cartilage differentiation. It can be seen that the cells do not differentiate. Each test result will be specifically described below. FIG. 6 shows the result of bone differentiation, FIGS. 7 and 8 show the result of cartilage differentiation, and FIG. 9 shows the result of fat differentiation. In each figure, the symbol shown on the horizontal axis indicates the number of the cell population, the parameter + indicates the cell population on which differentiation was induced, and-indicates the cell population on which differentiation was not induced. M 1 to M 8 indicate cell population symbols of mesenchymal stem cells (population) obtained from several persons.

図６によると、骨分化誘導を行った細胞集団は骨分化誘導を行わなかった細胞集団よりも1μgDNA当たりのカルシウム量μgがいずれも10倍以上多く、各細胞集団は骨分化したことが分かる。さらに、アリザリン赤による染色試験においても、すべての細胞集団が十分に染色されており、骨分化されていることを確認した。すなわち、各細胞集団は骨分化能を有し、骨分化誘導を行わないと骨分化しないことが分かる。   According to FIG. 6, the cell population in which bone differentiation was induced was 10 times or more higher in calcium content per 1 μg DNA than the cell population in which bone differentiation was not induced, indicating that each cell population had undergone bone differentiation. Furthermore, in the staining test with alizarin red, it was confirmed that all cell populations were sufficiently stained and differentiated into bone. That is, it can be seen that each cell population has a bone differentiation ability and does not undergo bone differentiation unless bone differentiation induction is performed.

また、図７に示すように、アルカリフォスファターゼ活性についても同様に、骨分化誘導を行った細胞集団の1min、1μgDNA当たりのアルカリフォスファターゼ活性は骨分化誘導を行わなかった細胞集団の数倍以上になっており、この結果から、各細胞集団は骨分化能を有し、骨分化誘導を行わないと骨分化しないことが分かる。   In addition, as shown in FIG. 7, the alkaline phosphatase activity of the cell population in which bone differentiation was induced was 1 min, and the alkaline phosphatase activity per 1 μg DNA was more than several times that of the cell population in which bone differentiation was not induced. From this result, it can be seen that each cell population has the ability to differentiate into bone and does not differentiate into bone unless bone differentiation is induced.

図８によると、軟骨分化誘導を行った細胞集団は軟骨分化誘導を行わなかった細胞集団よりも1μgDNA当たりのグリコサミノグルカン量μgがいずれも数倍以上多く、各細胞集団は軟骨分可能を有し、軟骨分化誘導を行わない限り軟骨分化をしないことが分かる。   According to FIG. 8, the cell population in which cartilage differentiation is induced has a glycosaminoglucan content μg per 1 μg DNA several times higher than the cell population in which cartilage differentiation is not induced. It can be seen that cartilage differentiation is not performed unless cartilage differentiation is induced.

また、図９によると、脂肪分化誘導を行った細胞集団は脂肪分化誘導を行わなかった細胞集団よりもOil-red-O（オイルレッドＯ）の吸光度540nmによる分光分析の強度は数倍以上になっており、各細胞集団は脂肪分化したことが分かる。そして、各細胞集団は脂肪分化能を有し、脂肪分化誘導を行わないと脂肪分化しないことが分かる。   Further, according to FIG. 9, the cell population in which fat differentiation was induced was more than several times stronger than the cell population in which fat differentiation was not induced, by the spectral analysis using Oil-red-O (oil red O) absorbance at 540 nm. It can be seen that each cell population has undergone fat differentiation. And each cell population has fat differentiation ability, and it turns out that fat differentiation does not occur unless fat differentiation induction is performed.

なお、図１の遺伝子発現データは、腸骨由来の骨髄から採取した間葉系幹細胞を用いて以下に示す方法により求めた。まず、骨髄の採取方法は、シリンジとニードルを用いて骨髄を吸引・採取し、等量の200U/mLのヘパリンナトリウムを含む基本培地に添加し、転倒混和し、これを骨髄細胞液として常温にて保管した。基本培地は、DMEM（Sigma D6046相当品）、10%FBS、Penicillin-Streptomycin（Sigma P0781相当品）を用いた。   In addition, the gene expression data of FIG. 1 was calculated | required by the method shown below using the mesenchymal stem cell extract | collected from the bone marrow derived from an iliac bone. First, the bone marrow is collected by aspirating and collecting the bone marrow using a syringe and needle, adding it to a basic medium containing an equal volume of 200 U / mL heparin sodium, mixing by inversion, and using this as a bone marrow cell solution at room temperature. And stored. As a basic medium, DMEM (Sigma D6046 equivalent), 10% FBS, and Penicillin-Streptomycin (Sigma P0781 equivalent) were used.

初代培養方法は、以下のように行った。すなわち、まず、骨髄細胞液に、クリーンベンチ内で適量の培地を加え、白血球数(WBC)と赤球数(RBC)を測定した。本例のように、腸骨由来の骨髄を用いる場合は、WBC/RBCの百分率が0.5%以上で、1mLの骨髄液当たりWBCが1000万個含まれていることが望ましい。つぎに、遠心し、遠心沈査に適量の培地を加えて培養皿(CORNING)に、WBCが1x105cells/cm2以上となるように播種し、37℃、5%CO2条件下で培養した。播種した3日後に浮遊細胞を除去し、新鮮な基本培地に交換した。その後、接着した細胞に1ng/mLのFGF-2を添加した。FGF-2は１日おきに添加した。 The primary culture method was performed as follows. That is, first, an appropriate amount of medium was added to the bone marrow cell solution in a clean bench, and the white blood cell count (WBC) and red cell count (RBC) were measured. As in this example, when bone marrow derived from the iliac bone is used, it is desirable that the percentage of WBC / RBC is 0.5% or more and 10 million WBC is contained per 1 mL of bone marrow fluid. Next, centrifuge, add an appropriate amount of medium to the centrifugal sedimentation, inoculate the culture dish (CORNING) so that the WBC is 1x10 5 cells / cm 2 or more, and cultured under conditions of 37 ° C, 5% CO 2 . Three days after seeding, floating cells were removed and replaced with fresh basal medium. Thereafter, 1 ng / mL FGF-2 was added to the adhered cells. FGF-2 was added every other day.

継代培養方法は、0.25% Trypsin、1mM EDTA・4Na (Gibco 25200-56 相当品)を用いて上記のように培養した細胞を培養皿から剥離させ、これに基本培地を適量添加した後、その細胞液を遠心力300gで遠心した。その後、遠心沈査に適量の基本培地を添加し、攪拌し、5000 個の細胞を100-mm培養皿(CORNING 430167相当品)に播種し、1ng/mLのFGF-2を含む10mLの基本培地で培養した。   The subculture method consists of detaching the cells cultured as described above using 0.25% Trypsin, 1 mM EDTA · 4Na (Gibco 25200-56 equivalent), and adding an appropriate amount of basal medium to the culture dish. The cell fluid was centrifuged at a centrifugal force of 300 g. Then add an appropriate amount of basal medium for centrifugation, stir, inoculate 5000 cells into a 100-mm culture dish (CORNING 430167 equivalent), and use 10 mL basal medium containing 1 ng / mL FGF-2. Cultured.

遺伝子の複製方法は、以下のように行った。すなわち、First-strand cDNAは、1μgの全RNAからReverTra Ace-alpha(Toyobo)を用いて合成した。合成したcDNAを鋳型として、ABI Prism 7700 Sequence Detection System instrument and software(PE Applied Biosystems Inc., Foster City CA)を用いてReal time PCRを行った。解析に用いた遺伝子のプライマーとプローブとしては、PE Applied Biosystemsから購入したTaqMan Gene Expression AssaysまたはGene Expression Micro Fluidic Cardを用いた。   The gene replication method was performed as follows. That is, First-strand cDNA was synthesized from 1 μg of total RNA using ReverTra Ace-alpha (Toyobo). Real time PCR was performed using the synthesized cDNA as a template and ABI Prism 7700 Sequence Detection System instrument and software (PE Applied Biosystems Inc., Foster City CA). TaqMan Gene Expression Assays or Gene Expression Micro Fluidic Card purchased from PE Applied Biosystems were used as gene primers and probes used in the analysis.

mRNA発現強度の測定は、各遺伝子について、ベータアクチンの遺伝子発現量で補正し、Comparative Ct法を用いて相対的なmRNAレベルを計算した。なお、統計解析は、Student's t-test を用いておこなった。   For the measurement of mRNA expression intensity, the relative mRNA level was calculated using the Comparative Ct method after correcting for the gene expression level of beta-actin for each gene. Statistical analysis was performed using Student's t-test.

図６〜９のデータは以下の方法により求めた。すなわち、カルシウム量は、Gitelmanらの方法(Anal Biochem 18:521-531 1967)に準じた方法を用いて測定した。グリコサミノグルカン量は、sulfated GAG assay kit(Biocolor)を用い、Farndale らの方法(Connect Tissue Res 9:247-248 1982)に準じた方法を用いて測定した。アルカリフォスファターゼ活性は、Besseyらの方法(J Biol Chem 164:321-329 1946)に準じた方法を用いて測定した。オイルレッドOの相対強度の測定は、12wellプレートで培養した細胞に1well当たり1mLの0.3%オイルレッドO（WAKO154-02072）:水＝6：4に混合した溶液を添加し、37℃で15 分間染色した後、1well当たり4mLの水で3回洗浄し、これを1well当たり1mLのイソプロパノールを加えて30分間色素を溶出させ、その溶出液を分光光度計で波長540nmについて測定した。DNA量は、PicoGreen dsDNA Quantitation kit(Molecular Probes)を用いて測定した。   The data in FIGS. 6 to 9 were obtained by the following method. That is, the amount of calcium was measured using a method according to the method of Gitelman et al. (Anal Biochem 18: 521-531 1967). The amount of glycosaminoglucan was measured using a sulfated GAG assay kit (Biocolor) using a method according to the method of Farndale et al. (Connect Tissue Res 9: 247-248 1982). Alkaline phosphatase activity was measured using a method according to the method of Bessey et al. (J Biol Chem 164: 321-329 1946). To measure the relative strength of Oil Red O, add 1 mL of 0.3% Oil Red O (WAKO154-02072): water = 6: 4 mixed solution to cells cultured in a 12-well plate, and continue at 37 ° C for 15 minutes. After staining, the plate was washed 3 times with 4 mL of water per well, 1 mL of isopropanol per well was added to elute the dye for 30 minutes, and the eluate was measured with a spectrophotometer at a wavelength of 540 nm. The amount of DNA was measured using PicoGreen dsDNA Quantitation kit (Molecular Probes).

以上本発明に係る間葉系幹細胞の均質性識別方法について説明した。しかしながら、均質性識別の基準となる基準間葉系幹細胞は必ずしもクローン、あるいはクローンと同等な均質性を有する細胞集団でなくても良い。例えば、複数の異なる他人から得た間葉系幹細胞であっても良い。   The method for identifying homogeneity of mesenchymal stem cells according to the present invention has been described above. However, the reference mesenchymal stem cells that serve as a reference for homogeneity identification are not necessarily clones or cell populations having homogeneity equivalent to clones. For example, mesenchymal stem cells obtained from a plurality of different others may be used.

図１０は、異なる４人P1〜P4から得た腸骨由来の間葉系幹細胞の1〜4回の継代培養された細胞集団に関し、図１と同様な方法で求めた遺伝子発現パターンである。図１１は、図１０のP1〜P4についての遺伝子のmRNA発現強度について最も高い強度を示す直線を連結した上限線と、最も低い強度を示す直線を連結した下限線とで囲まれる範囲を斜線部で表し、図１の母集団の遺伝子発現パターンを破線で併記したグラフである。   FIG. 10 is a gene expression pattern obtained by the same method as in FIG. 1 for a cell population that has been subcultured 1 to 4 times from iliac-derived mesenchymal stem cells obtained from four different persons P1 to P4. . FIG. 11 shows the range surrounded by the upper limit line connecting the straight lines showing the highest intensity and the lower limit line connecting the straight lines showing the lowest intensity for the mRNA expression intensity of the genes for P1 to P4 in FIG. 2 is a graph in which the gene expression pattern of the population of FIG.

図１０において、横軸は遺伝子番号を示し、縦軸はmRNA発現強度を示す。遺伝子番号は、図２に示す遺伝子を示す。パラメータP1〜P4は異なる4人の骨髄間葉系幹細胞（母集団）のそれぞれの遺伝子発現パターンを示し、F1〜F4は図１の線維芽細胞の遺伝子発現パターンを併記したものである。図１０によると、全体的に見ればP1〜P4の遺伝子発現パターンは図１のクローンC1〜C8の遺伝子発現パターンとよく類似していることが分かる。また、P1〜P4の各遺伝子のmRNA発現強度は、線維芽細胞のmRNA発現強度とは強度レベルが異なり明確に区別されていることが分かる。 In FIG. 10, the horizontal axis indicates the gene number, and the vertical axis indicates the mRNA expression intensity. The gene number indicates the gene shown in FIG. Parameters P 1 to P 4 indicate the gene expression patterns of four different bone marrow mesenchymal stem cells (population), and F 1 to F 4 indicate the gene expression patterns of fibroblasts in FIG. is there. According to FIG. 10, it can be seen that the gene expression patterns of P 1 to P 4 are generally similar to the gene expression patterns of clones C 1 to C 8 in FIG. Also, mRNA expression intensity of each gene P 1 to P 4 is seen to be clearly distinguished different intensity levels and mRNA expression level of fibroblasts.

さらに、図１１によると、図１０と図１は類似度が高いことが分かる。すなわち、図１１と図５は類似しており、しかも、図１１において破線で示される図１の母集団の遺伝子パターンがP1〜P4の遺伝子発現の上限線と下限線に囲まれる斜線部によく含まれていることが分かる。さらに、図５の場合と同様に、図１１において破線を上方に移動すればその大部分が斜線部に含まれるようになることも分かる。 Further, according to FIG. 11, it can be seen that FIG. 10 and FIG. That is, FIG. 11 and FIG. 5 are similar, and the gene pattern of the population of FIG. 1 indicated by a broken line in FIG. 11 is a hatched portion surrounded by the upper limit line and lower limit line of the gene expression of P 1 to P 4. It can be seen that it is often included. Further, as in FIG. 5, it can also be seen that if the broken line is moved upward in FIG. 11, most of the broken line is included in the hatched portion.

このように、判別を行おうとする間葉系幹細胞においてその均質性を判断するための基準となる多機能間葉系幹細胞は、クローンに限らずその母集団あるいは異なる人から採取された多機能間葉系幹細胞であっても良いことが分かる。   In this way, the multi-functional mesenchymal stem cells that serve as a standard for determining the homogeneity of the mesenchymal stem cells to be distinguished are not limited to clones, but between multi-functional samples collected from the population or from different people. It turns out that it may be a leaf stem cell.

以上説明したように、図式化された遺伝子発現パターンの類似性を判断することにより、間葉系幹細胞の同質性又は均質性を判別することができる。この遺伝子発現パターンの類似性を判別するために他の図式化を用いることができる。例えば、図１２に示す棒グラフ表示を用いることができる。図１２は、図１０に示すP1〜P4ついてのmRNA発現強度を遺伝子ごとに棒グラフで表したものである。 As described above, the homogeneity or homogeneity of mesenchymal stem cells can be determined by determining the similarity of the schematized gene expression pattern. Other schemes can be used to determine the similarity of this gene expression pattern. For example, the bar graph display shown in FIG. 12 can be used. FIG. 12 is a bar graph showing the mRNA expression intensity for P 1 to P 4 shown in FIG. 10 for each gene.

図１２によると、各遺伝子についてのmRNA発現強度にばらつきが見られるが、そのばらつきは大きくなく、そのバラツキの平均値からみれば小さいことが分かる。すなわち、例えば、予め均一な細胞集団から求められた遺伝子発現強度の平均値を基準とし、その平均値と判別しようとする間葉系幹細胞の遺伝子のmRNA発現強度との分散和が、所定の範囲内にあるならば、その判別しようとする間葉系幹細胞は、均質な多機能間葉系幹細胞であるとすることができる。なお、上記において、平均値として単純平均値を用いることができるが、必ずしも単純平均値でなくてもよい。所定の加重平均値を用いることができ、また、クローンの遺伝子が発現するmRNA発現強度を基準とすることもできる。   According to FIG. 12, it can be seen that there is a variation in the mRNA expression intensity for each gene, but the variation is not large and is small when viewed from the average value of the variation. That is, for example, based on the average value of gene expression strength obtained in advance from a uniform cell population, the dispersion sum of the average value and the mRNA expression strength of the gene of the mesenchymal stem cell to be distinguished is within a predetermined range. The mesenchymal stem cell to be identified can be a homogeneous multifunctional mesenchymal stem cell. In the above description, a simple average value can be used as the average value, but it is not necessarily required to be a simple average value. A predetermined weighted average value can be used, and the mRNA expression intensity at which a clone gene is expressed can be used as a reference.

継代培養された間葉系幹細胞の遺伝子群に関するmRNA発現強度を図式化したグラフである。It is the graph which expressed the mRNA expression intensity about the gene group of the mesenchymal stem cell by which subculture was carried out. 図１に遺伝子番号で示す記号の遺伝子略称、名称及びGenBank 登録番号を示す説明図である。It is explanatory drawing which shows the gene abbreviation of the symbol shown by the gene number in FIG. 1, a name, and a GenBank registration number. 図２の遺伝子に準ずる遺伝子に関する遺伝子略称、名称及びGenBank 登録番号を示す説明図である。It is explanatory drawing which shows the gene abbreviation regarding the gene according to the gene of FIG. 2, a name, and a GenBank registration number. 図２の遺伝子に準ずる遺伝子に関する遺伝子略称、名称及びGenBank 登録番号を示す説明図である。It is explanatory drawing which shows the gene abbreviation regarding the gene according to the gene of FIG. 2, a name, and a GenBank registration number. 図１のグラフを加工した図である。It is the figure which processed the graph of FIG. 数人の間葉系幹細胞（母集団）の骨分化能を示すグラフである。It is a graph which shows the bone differentiation ability of several mesenchymal stem cells (population). 数人の間葉系幹細胞（母集団）の骨分化能を示すグラフである。It is a graph which shows the bone differentiation ability of several mesenchymal stem cells (population). 数人の間葉系幹細胞（母集団）の軟骨分化能を示すグラフである。It is a graph which shows the cartilage differentiation ability of several mesenchymal stem cells (population). 数人の間葉系幹細胞（母集団）の脂肪分化能を示すグラフである。It is a graph which shows the fat differentiation ability of several mesenchymal stem cells (population). 異なる4人から得た間葉系幹細胞を継代培養し、その細胞集団の遺伝子群に関するmRNA発現強度を図式化したグラフである。It is the graph which subcultured the mesenchymal stem cell obtained from four different persons, and schematized the mRNA expression intensity regarding the gene group of the cell population. 図１０グラフを加工した図である。It is the figure which processed the FIG. 10 graph. 図１０の４人に関するmRNA発現強度を棒グラフ表示した図である。It is the figure which displayed the mRNA expression intensity regarding four persons of FIG. 10 with the bar graph.

Claims (7)

継代培養された間葉系幹細胞の細胞集団から複数のクローンを作製する段階と、該クローン及びその母集団について所定の遺伝子群に関するmRNA発現強度を取得する段階と、該取得された前記クローン及びその母集団のmRNA発現強度群を前記遺伝子群に対して図式化した遺伝子発現パターンを求める段階と、該得られた遺伝子発現パターンから前記母集団の均質性を判別する段階と、からなる間葉系幹細胞の均質性識別方法。 A method of making a multiple clones from the cell population of mesenchymal stem cells are subcultured, the steps of obtaining the mRNA expression level for a given gene cluster for the clone and its population, the clones and was the acquired A mesenchyme comprising a step of obtaining a gene expression pattern in which the mRNA expression intensity group of the population is diagrammed with respect to the gene group, and a step of determining the homogeneity of the population from the obtained gene expression pattern A method for identifying homogeneity of stem cells. 継代培養された間葉系幹細胞の所定の遺伝子群に関するmRNA発現強度を取得する段階と、該取得されたmRNA発現強度群を前記遺伝子群に対して図式化した遺伝子発現パターンを求める段階と、該遺伝子発現パターンから前記細胞集団の均質性を判別する段階と、からなる間葉系幹細胞の均質性識別方法。   Obtaining a mRNA expression intensity for a predetermined gene group of subcultured mesenchymal stem cells; obtaining a gene expression pattern in which the obtained mRNA expression intensity group is schematized with respect to the gene group; and Determining the homogeneity of the cell population from the gene expression pattern, and a method for identifying the homogeneity of mesenchymal stem cells. 請求項１または２に記載の方法により識別された均質間葉系幹細胞。   Homogeneous mesenchymal stem cells identified by the method according to claim 1 or 2. 間葉系幹細胞の細胞集団から作製されたクローンであって、脂肪分化、骨分化及び軟骨分化の３つの方向に分化する分化能を有し、分化誘導しない限りいずれの分化もしない基準間葉系幹細胞から得られる所定の遺伝子群に関する遺伝子発現パターンに類似する遺伝子発現パターンを有する均質間葉系幹細胞。 A clone prepared from a cell population of mesenchymal stem cells, having a differentiation ability to differentiate in three directions of adipose differentiation, bone differentiation, and cartilage differentiation, and not differentiating unless differentiation is induced A homogeneous mesenchymal stem cell having a gene expression pattern similar to a gene expression pattern for a predetermined gene group obtained from a stem cell. 所定の遺伝子群に関する各mRNA発現強度と、該遺伝子群に関し間葉系幹細胞の細胞集団から作製されたクローンであって、脂肪分化、骨分化及び軟骨分化の３つの方向に分化する分化能を有し、分化誘導しない限りいずれの分化もしない基準間葉系幹細胞から得られる各mRNA発現強度との分散和をとった場合に、その分散和が所定の範囲内にある均質間葉系幹細胞。 A clone prepared from a cell population of mesenchymal stem cells related to a specific gene group and a differentiation ability to differentiate in three directions of adipose differentiation, bone differentiation and cartilage differentiation. A homogeneous mesenchymal stem cell in which the dispersion sum is within a predetermined range when the dispersion sum is taken with each mRNA expression intensity obtained from a reference mesenchymal stem cell that does not undergo any differentiation unless differentiation is induced. 所定の遺伝子群は、CTGF、IGFBP7、KCTD12、LAMA3、LIF、MGP、PRG1、TRIB2、IGF1、BMP4、SERPINI1、CD74、HLA-DRA、HLA-DRB3、TGM2、DNCI1、MCAM、TFPI2、ARHGDIBのいずれか複数の遺伝子からなる遺伝子群であることを特徴とする請求項１〜５のいずれかに記載の間葉系幹細胞の均質性識別方法又は均質間葉系幹細胞。   The given gene group is CTGF, IGFBP7, KCTD12, LAMA3, LIF, MGP, PRG1, TRIB2, IGF1, BMP4, SERPINI1, CD74, HLA-DRA, HLA-DRB3, TGM2, DNCI1, MCAM, TFPI2, ARHGDIB The method for identifying homogeneity of mesenchymal stem cells or homogeneous mesenchymal stem cells according to any one of claims 1 to 5, wherein the gene group is composed of a plurality of genes. 所定の遺伝子群は、ADAMTS1、ABHD2、ADD3、ANXA10、ACLY、ATP6V1G3、BDNF、BRIP1、CDH6、CPA4、CDC25A、CHI3L1、F2R、F2RL1、CCND1、CKAP2、DNAH3、SMURF2、EFEMP1、DCBLD、ESM1、EDG2、LOC221810、GABRB1、GATA6、GMFG、HGF、HMGA2、PHLDB2、HTR7、MGC14161、DGKG、PRDM16、MCTP2、FLJ23033、FLJ35681、FLJ38725、C9orf72、LYPDC1、IF、IGFBP1、IGFBP3、IGFBP5、ITGA5、ITGB3、IFI30、IL6、KRT19、KRT23、KRTAP1-5、KRTHA4、KCTD16、KIAA1913、LXN、LEPR、Lrp2bp、MET、MICA、LF1、PR3、TN4、ASK、LAU、CTD4、4HA2、TGER1、ROS1、AB27B、AC2、GS4、ARRES1、LEKHK1、YPN、LC16A4、LC2A1、LC20A1、AMD3、UHW2、YT1、RPC4、CHL1のいずれか複数の遺伝子からなる遺伝子群であることを特徴とする請求項１〜５のいずれかに記載の間葉系幹細胞の均質性識別方法又は均質間葉系幹細胞。   The predetermined gene groups are ADAMTS1, ABHD2, ADD3, ANXA10, ACLY, ATP6V1G3, BDNF, BRIP1, CDH6, CPA4, CDC25A, CHI3L1, F2R, F2RL1, CCND1, CKAP2, DNAH3, SMURF2, EFEMP1, DCBED, M LOC221810, GABRB1, GATA6, GMFG, HGF, HMGA2, PHLDB2, HTR7, MGC14161, DGKG, PRDM16, MCTP2, FLJ23033, FLJ35681, FLJ38725, C9orf72, LYPDC1, IF, IGFBP1, IGFBP3, IGFBP3, ITGAIF5, ITGAIF3 KRT19, KRT23, KRTAP1-5, KRTHA4, KCTD16, KIAA1913, LXN, LEPR, Lrp2bp, MET, MICA, LF1, PR3, TN4, ASK, LAU, CTD4, 4HA2, TGER1, ROS1, AB27B, AC2, GS4, ARRES1, The mesenchymal system according to any one of claims 1 to 5, wherein the mesenchymal system is a gene group consisting of a plurality of genes of LEKHK1, YPN, LC16A4, LC2A1, LC20A1, AMD3, UHW2, YT1, RPC4, and CHL1. A method for identifying homogeneity of stem cells or homogeneous mesenchymal stem cells.
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