JP6419455B2 - Method for producing mature mast cells and obtained mature mast cells - Google Patents
Method for producing mature mast cells and obtained mature mast cells Download PDFInfo
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- JP6419455B2 JP6419455B2 JP2014100469A JP2014100469A JP6419455B2 JP 6419455 B2 JP6419455 B2 JP 6419455B2 JP 2014100469 A JP2014100469 A JP 2014100469A JP 2014100469 A JP2014100469 A JP 2014100469A JP 6419455 B2 JP6419455 B2 JP 6419455B2
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Description
本発明は、マスト細胞からの成熟マスト細胞の作製方法に関する。さらには、前記作製方法により作製された成熟マスト細胞に関する。 The present invention relates to a method for producing mature mast cells from mast cells. Furthermore, the present invention relates to a mature mast cell produced by the production method.
マスト細胞（mast cell）は、別名肥満細胞や顆粒細胞とも呼ばれ、哺乳類の粘膜下組織や結合組織などに存在する。マスト細胞は細胞内に顆粒を有し、顆粒は高度に硫酸化されたヘパリンの存在により、塩基性色素で強く染色される。生体内で形成されるマスト細胞は、他の血液細胞と同様に骨髄造血幹細胞由来である。また、好塩基性顆粒をもち血中に存在する好塩基球とは異なり、マスト細胞は末梢血中には成熟細胞としては存在しない。前駆マスト細胞として循環血中を経由した後、浸潤した組織において最終的な分化（成熟化）を遂げ、機能を発揮する。 Mast cells, also called mast cells and granule cells, are present in mammalian submucosa and connective tissues. Mast cells have granules in the cells, and the granules are strongly stained with a basic dye due to the presence of highly sulfated heparin. Mast cells formed in vivo are derived from bone marrow hematopoietic stem cells like other blood cells. In addition, unlike basophils present in blood with basophil granules, mast cells do not exist as mature cells in peripheral blood. After passing through the circulating blood as a precursor mast cell, it undergoes final differentiation (maturation) in the infiltrated tissue and exerts its function.
マスト細胞はIgEを介したI型アレルギー反応の主体である。マスト細胞の顆粒内にはヒスタミンをはじめとした各種化学伝達物質（ケミカルメディエーター）があり、細胞表面に結合したIgEに抗原が結合しその架橋が成立すると、それがトリガーとなって細胞膜酵素の活性化がうながされ、結果的に顆粒内容物である特異顆粒、すなわちヒスタミンなどが放出される（脱顆粒）。また、細胞膜酵素の活性化は、アラキドン酸の生成と代謝を亢進させ（アラキドン酸カスケード）、代謝物であるロイコトリエン、血小板活性化因子（PAF）、プロスタグランジン、トロンボキサンA2などを細胞膜から遊離する。こうしたマスト細胞から遊離されたケミカルメディエーターのうち、ヒスタミンやロイコトリエンC4などは気管支平滑筋収縮作用、血管透過性亢進作用、粘液分泌作用などを有し、アレルギーにおける即時型反応を引き起こす。一方、血小板活性化因子やロイコトリエンB4などは遊走因子として好酸球や好中球などの炎症細胞を反応局所に呼び寄せる。これはアレルギーの遅延型反応（アレルギー性炎症）を引き起こす。 Mast cells are the main component of type I allergic reaction via IgE. There are various chemical mediators such as histamine in the granule of mast cells. When the antigen binds to IgE bound to the cell surface and the cross-linking is established, the activity of cell membrane enzyme is triggered. As a result, specific granules which are granule contents, that is, histamine and the like are released (degranulation). In addition, activation of cell membrane enzymes enhances arachidonic acid production and metabolism (arachidonic acid cascade), releasing metabolites leukotrienes, platelet activating factor (PAF), prostaglandins, thromboxane A2, etc. from cell membranes. To do. Among the chemical mediators released from such mast cells, histamine, leukotriene C4, etc. have bronchial smooth muscle contraction action, vascular permeability enhancement action, mucus secretion action, etc., and cause an immediate reaction in allergy. On the other hand, platelet activating factor, leukotriene B4, and the like attract inflammatory cells such as eosinophils and neutrophils as migratory factors to the reaction site. This causes a delayed allergic reaction (allergic inflammation).
骨髄由来マスト細胞（bone marrow-derived mast cell：BMMC）を線維芽細胞株と幹細胞増殖因子（stem cell factor：SCF） 存在下で共培養することにより、成熟マスト細胞である結合組織型マスト細胞（connective tissue type mast cell：CTMC）様の培養マスト細胞が得られることが報告されている（非特許文献１）。ここでは、フィーダー細胞であるSwiss 3T3線維芽細胞株をマイトマイシンCで処理することにより、BMMCからCTMC様細胞への分化を進展させうる共培養系を確立することに成功したことが報告されている。また、BMMCを線維芽細胞株とヒアルロン酸量が低下した条件下で共培養することにより、成熟マスト細胞が得られることが報告されている（特許文献１）。マウスiPS細胞由来マスト細胞をOP9骨髄ストローマ細胞株（正常なM-CSF遺伝子を欠損したop/opマウスに由来する骨髄ストローマ細胞株）と共に培養することで、CTMC様の培養マスト細胞を得ることができたことが報告されている（非特許文献２）。 Bone marrow-derived mast cells (BMMC) are co-cultured in the presence of a fibroblast cell line and a stem cell factor (SCF), so that connective tissue mast cells (mast mast cells) It has been reported that cultured mast cells like connective tissue type mast cells (CTMC) can be obtained (Non-patent Document 1). Here, it has been reported that the Swiss 3T3 fibroblast cell line, a feeder cell, was successfully treated with mitomycin C to establish a co-culture system that can promote differentiation from BMMC to CTMC-like cells. . In addition, it has been reported that mature mast cells can be obtained by co-culturing BMMC with fibroblast cell lines under conditions where the amount of hyaluronic acid is reduced (Patent Document 1). It is possible to obtain CTMC-like cultured mast cells by culturing mouse iPS cell-derived mast cells with OP9 bone marrow stromal cell line (bone marrow stromal cell line derived from op / op mice deficient in normal M-CSF gene) It has been reported that it was made (Non-patent Document 2).
上記で説明したように成熟マスト細胞は浸潤した組織において最終的な分化・成熟化を遂げるため、成熟マスト細胞を回収するのは困難である。効果的に成熟マスト細胞を作製することで、未だ明らかにされていないマスト細胞の末梢組織での成熟化メカニズムなどの解明が期待される。そこで、より効果的に成熟マスト細胞を作製するための開発が望まれている。 As explained above, mature mast cells undergo final differentiation and maturation in the infiltrated tissue, and it is difficult to recover mature mast cells. By effectively producing mature mast cells, elucidation of the maturation mechanism of mast cells in peripheral tissues that has not yet been clarified is expected. Therefore, development for producing mature mast cells more effectively is desired.
Wntファミリーは分泌型の糖タンパク質で、細胞生命の調節や様々な発生、発癌などに関わっているといわれている。Wntシグナルは生物種を超えて保存されており、発生初期における体軸形成や器官形成、細胞の増殖や分化を制御している。Wntシグナル伝達経路には、１）β-カテニンの細胞内蓄積を介して転写因子を制御するβ-カテニン経路、２）低分子量Ｇタンパク質であるRhoファミリーを介して平面内細胞極性(planar cell polarity)を制御するPCP経路、３）３量体Ｇタンパク質を介して細胞内でCa2+の動員を引き起こし、プロテインキナーゼＣ (PKC; protein kunase C)やCaMK II (Ca2+/calmodulin-dependent protein kinase II)などを活性化するCa2+経路の３種類が少なくとも存在することが公知である（蛋白質核酸酵素, Vol.49(10), 1421-1427 (2004)）。また、Wntシグナルネットワークとその異常による病態についても報告がある（生化学, Vol.81(9), 780-792 (2009)）。これらの文献には、Wntシグナル経路の多様性については示されているものの、Wntシグナル経路とマスト細胞分化との関係については、一切記載も示唆もされていない。 The Wnt family is a secreted glycoprotein and is said to be involved in the regulation of cell life, various developments, and carcinogenesis. The Wnt signal is conserved across species and controls body axis formation, organogenesis, cell proliferation and differentiation in the early stages of development. The Wnt signaling pathway includes 1) β-catenin pathway that regulates transcription factors through intracellular accumulation of β-catenin, and 2) planar cell polarity via Rho family, which is a low molecular weight G protein. 3) PCP pathway that regulates 3) Intrinsic Ca 2+ mobilization via trimeric G protein, protein kinase C (PKC; protein kunase C) and CaMK II (Ca 2+ / calmodulin-dependent protein) It is known that there are at least three types of Ca 2+ pathways that activate kinase II) and the like (protein nucleic acid enzyme, Vol. 49 (10), 1421-1427 (2004)). There are also reports on the Wnt signal network and its pathological conditions (Biochemistry, Vol. 81 (9), 780-792 (2009)). Although these documents show the diversity of the Wnt signaling pathway, there is no description or suggestion of the relationship between the Wnt signaling pathway and mast cell differentiation.
本発明は、細胞培養系においてマスト細胞から成熟マスト細胞の作製方法を提供することを課題とする。さらには、当該作製方法によって作製された成熟マスト細胞を提供することを課題とする。 An object of the present invention is to provide a method for producing mature mast cells from mast cells in a cell culture system. Furthermore, it aims at providing the mature mast cell produced by the said production method.
本発明者等は、骨髄由来マスト細胞をSwiss 3T3細胞株又はOP9細胞株と共に培養することで、結合組織型マスト細胞様の成熟マスト細胞を得ることができたことに着目し、上記課題を達成するために鋭意研究を重ねた結果、マスト細胞の成熟化メカニズムにWntシグナルが関与していることを初めて見出し、本発明を完成した。また、ヒト多能性幹細胞由来のマスト細胞を、線維芽細胞と共培養することにより、成熟マスト細胞を得ることができたことに基づき、本発明を完成した。 The inventors of the present invention have achieved the above-mentioned problem by paying attention to the fact that connective tissue type mast cell-like mature mast cells could be obtained by culturing bone marrow-derived mast cells with the Swiss 3T3 cell line or OP9 cell line. As a result of intensive studies, the inventors found for the first time that the Wnt signal is involved in the maturation mechanism of mast cells and completed the present invention. Further, the present invention was completed based on the fact that mature mast cells could be obtained by co-culturing mast cells derived from human pluripotent stem cells with fibroblasts.
即ち本発明は、以下よりなる。
１．マスト細胞をWntで刺激することを特徴とする成熟マスト細胞の作製方法。
２．マスト細胞が、人工多能性幹細胞、採取された胚性幹細胞及び造血幹細胞から選択されるいずれかの細胞由来のマスト細胞である、前項１に記載の作製方法。
３．マスト細胞をWntで刺激することが、1×105〜5×105 cells/mlのマスト細胞を、Wntが5〜100 ng/ml含まれる培地中で培養することによる、前項１又は２に記載の作製方法。
４．培地に、更に幹細胞増殖因子及び／又はインターロイキン３を含む、前項３に記載の作製方法。
５．WntがWnt5aである、前項１〜４のいずれか１に記載の作製方法。
６．ヒト多能性幹細胞由来のマスト細胞を、線維芽細胞と共培養することを特徴とする、成熟マスト細胞の作製方法。
７．線維芽細胞が、Swiss 3T3線維芽細胞、間葉系幹細胞であるC3H10T1/2細胞、マウスプライマリー皮膚線維芽細胞（MDF）から選択される、前項６に記載の作製方法。
８．ヒト多能性幹細胞由来のマスト細胞と線維芽細胞の共培養が、幹細胞増殖因子（SCF）およびインターロイキン６（IL-6）を添加した培地を用いて行われる、前項６または７に記載の作製方法。
９．前項１〜８のいずれか１に記載の方法で作製されたマスト細胞。
That is, this invention consists of the following.
1. A method for producing a mature mast cell, which comprises stimulating mast cells with Wnt.
2. 2. The production method according to item 1 above, wherein the mast cell is a mast cell derived from any cell selected from induced pluripotent stem cells, collected embryonic stem cells and hematopoietic stem cells.
3. Stimulating mast cells with Wnt is as described in 1 or 2 above by culturing mast cells of 1 × 10 5 to 5 × 10 5 cells / ml in a medium containing 5 to 100 ng / ml of Wnt. The manufacturing method as described.
4). 4. The production method according to item 3 above, wherein the medium further contains stem cell growth factor and / or interleukin 3.
5. 5. The production method according to any one of items 1 to 4, wherein Wnt is Wnt5a.
6). A method for producing mature mast cells, comprising co-culturing mast cells derived from human pluripotent stem cells with fibroblasts.
7). 7. The production method according to item 6 above, wherein the fibroblast is selected from Swiss 3T3 fibroblast, C3H10T1 / 2 cell that is a mesenchymal stem cell, and mouse primary skin fibroblast (MDF).
8). 8. The co-culture of human pluripotent stem cell-derived mast cells and fibroblasts is performed using a medium supplemented with stem cell growth factor (SCF) and interleukin 6 (IL-6), Manufacturing method.
9. A mast cell produced by the method according to any one of 1 to 8 above.
本発明の方法によれば、成熟マスト細胞を作製することができる。本発明の方法で作製された成熟マスト細胞によれば、生体内のマスト細胞の機能の理解が一段と深まることが期待される。さらに、マスト細胞の機能に着目した医薬品の開発に関しても貢献しうるものと期待される。 According to the method of the present invention, mature mast cells can be prepared. According to the mature mast cell produced by the method of the present invention, it is expected that the understanding of the function of the mast cell in the living body will be further deepened. Furthermore, it is expected to contribute to the development of pharmaceuticals that focus on the function of mast cells.
本明細書において「マスト細胞」とは、人工多能性幹細胞（iPS細胞: induced pluripotent stem cells）、採取された胚性幹細胞（ES細胞: embryonic stem cells）及び造血幹細胞から選択されるいずれの多能性幹細胞（PSC: pluripotent stem cells）由来であってもよい。背景技術の欄でも示したように、生体内で形成されるマスト細胞は骨髄造血幹細胞由来であるが、本発明の成熟マスト細胞の作製方法は、造血幹細胞の他、iPS細胞や採取されたES細胞由来のいずれのマスト細胞にも適用することができる。 As used herein, “mast cell” refers to any multiplicity selected from induced pluripotent stem cells (iPS cells), harvested embryonic stem cells (ES cells) and hematopoietic stem cells. It may be derived from pluripotent stem cells (PSC). As shown in the background section, mast cells formed in vivo are derived from bone marrow hematopoietic stem cells, but the method for producing mature mast cells of the present invention is not limited to hematopoietic stem cells, iPS cells, or collected ES cells. It can be applied to any mast cell derived from a cell.
iPS細胞とは、体細胞へ数種類の遺伝子を導入することにより、受精卵、余剰胚やES細胞を利用せずに分化細胞の初期化を誘導し、ES細胞と同様な多能性や増殖能を有する誘導多能性幹細胞をいい、2006年にマウスの線維芽細胞から世界で初めて作られた。さらに、マウスiPS細胞の樹立に用いた４遺伝子のヒト相同遺伝子であるOCT3/4、SOX2、KLF4、C-MYCを、ヒト由来線維芽細胞に導入してヒトiPS細胞の樹立に成功したことが報告されている(Cell 131: 861-872, 2007)。本発明で使用されるiPS細胞は、上記のような自体公知の方法により作製されたiPS細胞、又は今後開発される新たな方法により作製されるiPS細胞であってもよい。 iPS cells induce the reprogramming of differentiated cells without using fertilized eggs, surplus embryos or ES cells by introducing several types of genes into somatic cells. Induced pluripotent stem cells with, and were first made in 2006 from mouse fibroblasts in the world. Furthermore, we successfully established human iPS cells by introducing OCT3 / 4, SOX2, KLF4 and C-MYC, which are the four human homologous genes used to establish mouse iPS cells, into human fibroblasts. It has been reported (Cell 131: 861-872, 2007). The iPS cell used in the present invention may be an iPS cell produced by a method known per se as described above, or an iPS cell produced by a new method developed in the future.
ES細胞とは、一般的には胚盤胞期胚の内部にある内部細胞塊(inner cell mass)と呼ばれる細胞集塊をin vitro培養に移し、未分化幹細胞集団として単離した多能性幹細胞である。ES細胞は、M.J.Evans & M.H.Kaufman (Nature, 292, 154, 1981)に続いて、G.R.Martin (Natl.Acad.Sci.USA, 78, 7634, 1981)によりマウスで多分化能を有する細胞株として樹立された。ヒト由来ES細胞についても、既に多くの株が樹立されており、ES Cell International社、Wisconsin Alumni Research Foundation、National Stem Cell Bank (NSCB)等から入手することが可能である。ES細胞は、一般に初期胚を培養することにより樹立されるが、体細胞の核を核移植した初期胚からもES細胞を作製することが可能である。また、異種動物の卵細胞、又は脱核した卵細胞を複数に分割した細胞小胞(cytoplasts, ooplastoids)に、所望の動物の細胞核を移植して胚盤胞期胚様の細胞構造体を作製し、それを基にES細胞を作製する方法もある。また、単為発生胚を胚盤胞期と同等の段階まで発生させ、そこからES細胞を作製する試みや、ES細胞と体細胞を融合させることにより、体細胞核の遺伝情報を有したES細胞を作る方法も報告されている。本発明で使用されるES細胞は、上記のような自体公知の方法、又は今後開発される新たな方法により作製されるES細胞であってもよい。 ES cells are pluripotent stem cells that have been isolated as undifferentiated stem cell populations by transferring cell clusters called inner cell mass, which are generally inside blastocyst-stage embryos, to in vitro culture It is. ES cells are pluripotent cell lines in mice by MJEvans & MHKaufman (Nature, 292, 154, 1981) followed by GRMartin (Natl. Acad. Sci. USA, 78, 7634, 1981). Established. Many strains of human-derived ES cells have already been established and can be obtained from ES Cell International, Wisconsin Alumni Research Foundation, National Stem Cell Bank (NSCB), and the like. Although ES cells are generally established by culturing early embryos, ES cells can also be produced from early embryos obtained by nuclear transfer of somatic cell nuclei. In addition, the blastocyst stage embryo-like cell structure is prepared by transplanting the cell nucleus of the desired animal into a cell vesicle (cytoplasts, ooplastoids) obtained by dividing the egg cell of a heterogeneous animal or a denucleated egg cell into a plurality of cells, There is also a method for producing ES cells based on this. In addition, ES cells with genetic information on somatic cell nuclei by developing parthenogenetic embryos to the same stage as the blastocyst stage and trying to produce ES cells from them, or by fusing ES cells and somatic cells The method of making is also reported. The ES cell used in the present invention may be an ES cell produced by a method known per se as described above or a new method developed in the future.
本明細書の「マスト細胞」は、高親和性IgE 受容体であるFcεRIとSCFの受容体であるc-kit をともに発現する細胞として定義することができる。例えば、BMMCや幹細胞増殖因子（SCF）、インターロイキン３（IL-3）を用いて多能性幹細胞から誘導したマスト細胞が挙げられる。生体内では、染色性や刺激応答性に基づき、例えば、皮膚や腹腔などに分布する結合組織型マスト細胞（connective tissue-type mast cell, CTMC）や、粘膜型マスト細胞（mucosal mast cell, MMC）などの細胞が挙げられる。CTMCの分化は主としてSCF のシグナルに依存し、MMC の誘導にはIL-3やIL-10が関与しているといわれている。例えば、マウス骨髄細胞の場合、IL-3存在下で長期間培養することにより、ほぼ均一なマスト細胞の集団を得ることができる。これはIL-3依存性骨髄由来培養マスト細胞（BMMC）と呼ばれ、BMMCはc-kit とFcεRI の両方を発現し、IgEを介した抗原抗体反応により、脱顆粒、アラキドン酸代謝物産生、サイトカイン産生といった応答を示す。しかしながら、BMMC は、プロテオグリカンの成熟度や、ヒスタミン含量、顆粒内のプロテアーゼ発現、カチオン性刺激に対する脱顆粒応答といった指標等から、未成熟なマスト細胞の性質を反映する系と位置づけられている（生化学, Vol.82(11), 1021-1031 (2010)）。本明細書においてマスト細胞として、このようなBMMCを使用することができる。 A “mast cell” in the present specification can be defined as a cell that expresses both high affinity IgE receptor FcεRI and SCF receptor c-kit. For example, mast cells derived from pluripotent stem cells using BMMC, stem cell growth factor (SCF), or interleukin 3 (IL-3) can be mentioned. In vivo, for example, connective tissue-type mast cells (CTMC) or mucosal mast cells (MMC) distributed on the skin, abdominal cavity, etc. Cell. Differentiation of CTMC mainly depends on SCF signals, and it is said that IL-3 and IL-10 are involved in MMC induction. For example, in the case of mouse bone marrow cells, a substantially uniform population of mast cells can be obtained by culturing for a long time in the presence of IL-3. This is called IL-3-dependent bone marrow-derived cultured mast cells (BMMC). BMMC expresses both c-kit and FcεRI, and degranulation, arachidonic acid metabolite production, Responses such as cytokine production are shown. However, BMMC is positioned as a system that reflects the properties of immature mast cells based on proteoglycan maturity, histamine content, protease expression in granules, and degranulation response to cationic stimuli. Chemistry, Vol.82 (11), 1021-1031 (2010)). In this specification, such BMMC can be used as a mast cell.
本明細書において、その他のマスト細胞として、例えばiPS細胞やES細胞から自体公知の方法により胚様細胞塊を作製し、さらにSCF、IL-3やインターロイキン６（IL-6）を作用させて作製することができる。
ヒトiPS細胞またはヒトES細胞からマスト細胞を作製する場合は、まず、ヒトiPS細胞またはヒトES細胞から血液前駆細胞を分化誘導し、その後血液前駆細胞からマスト細胞を分化誘導して作製することができる。ヒトiPS細胞またはヒトES細胞から血液前駆細胞を分化誘導する方法は、例えばフィーダー細胞（好ましくはC3H10T1/2細胞）を予め播種した培養皿に、ヒトiPS細胞またはヒトES細胞を播種し、血管内皮細胞増殖因子（VEGF）含有培地中で15〜19日間培養する方法が例示される。またヒトiPSまたはヒトES細胞を、フィーダー細胞と、アクチビンA、骨形成因子（BMP）4、Rhoキナーゼ阻害剤（好ましくはY-27632）を含む培地中で1〜3日間培養し、その後BMP4、VEGFを含む培地中で1〜3日間培養し、BMP4、VEGF、TGF-β受容体阻害剤（好ましくはSB431542）を含む培地中で1〜3日間培養し、その後BMP4、VEGF、SCF、トロンボポエチン（TPO）を含む培地中で1〜4日間培養することにより、血液前駆細胞を分化誘導することができる。本明細書にて血液前駆細胞とは、細胞表面マーカーのCD34とCD43が陽性である細胞（CD34+CD43+細胞）を意味する。細胞表面マーカーは自体公知の手法により確認することができる。
次に、得られた血液前駆細胞をマスト細胞に分化誘導する。血液前駆細胞を、SCF、IL-6、IL-3を含む培地中にて12〜16日間培養し、その後SCF、IL-6を含む培地を添加して14〜30日間培養し、その後顕微鏡下でコロニーを採取し、当該コロニーをSCF、IL-6を含む培地中にて10〜20日間培養することによりマスト細胞を分化誘導することができる。
In the present specification, as other mast cells, for example, embryonic cell masses are prepared from iPS cells or ES cells by a method known per se, and SCF, IL-3 or interleukin 6 (IL-6) is further allowed to act. Can be produced.
When producing mast cells from human iPS cells or human ES cells, it is first possible to induce differentiation of blood progenitor cells from human iPS cells or human ES cells and then induce differentiation of mast cells from blood precursor cells. it can. A method for inducing differentiation of blood progenitor cells from human iPS cells or human ES cells is, for example, by seeding human iPS cells or human ES cells in a culture dish pre-seeded with feeder cells (preferably C3H10T1 / 2 cells), and then vascular endothelium. A method of culturing in a medium containing cell growth factor (VEGF) for 15 to 19 days is exemplified. Further, human iPS or human ES cells are cultured for 1 to 3 days in a medium containing feeder cells, activin A, bone morphogenetic factor (BMP) 4, Rho kinase inhibitor (preferably Y-27632), and then BMP4, Culturing in a medium containing VEGF for 1-3 days, culturing in a medium containing BMP4, VEGF, TGF-β receptor inhibitor (preferably SB431542) for 1-3 days, and then BMP4, VEGF, SCF, thrombopoietin ( Blood progenitor cells can be induced to differentiate by culturing in a medium containing TPO for 1 to 4 days. As used herein, blood progenitor cells mean cells that are positive for the cell surface markers CD34 and CD43 (CD34 + CD43 + cells). The cell surface marker can be confirmed by a method known per se.
Next, the obtained blood precursor cells are induced to differentiate into mast cells. Blood progenitor cells are cultured in a medium containing SCF, IL-6, IL-3 for 12 to 16 days, and then added with a medium containing SCF and IL-6, and cultured for 14 to 30 days. The mast cells can be induced to differentiate by collecting the colonies and culturing the colonies in a medium containing SCF and IL-6 for 10 to 20 days.
本発明においては、マスト細胞をWntで刺激することにより成熟マスト細胞を作製する。本明細書においてマスト細胞に作用して成熟マスト細胞に分化誘導しうるWntは、分化誘導可能なWntであればよく、Wntシグナル伝達経路についても、１）β-カテニン経路、２）PCP経路、３）Ca2+経路のいずれの経路を介するものであってもよい。本明細書において使用可能なWntとしては、Wnt5aが挙げられる。 In the present invention, mature mast cells are prepared by stimulating mast cells with Wnt. In this specification, Wnt capable of acting on mast cells and inducing differentiation into mature mast cells may be Wnt capable of inducing differentiation. Regarding Wnt signaling pathway, 1) β-catenin pathway, 2) PCP pathway, 3) It may be via any of the Ca 2+ pathways. Wnt that can be used herein includes Wnt5a.
本発明の成熟マスト細胞は、Wntでマスト細胞を刺激することで作製することができる。具体的には、上述したマスト細胞をWntを含む培地で培養することで成熟マスト細胞を作製することができる。より具体的には、マスト細胞の種類に応じて適宜調整可能であるが、5〜100 ng/ml、好ましくは20〜50 ng/mlのWntを含む培地に対して、1×105 cells/ml〜5×105 cells/mlの細胞を培養容器にて培養することにより達成される。マスト細胞の培養は、37℃において10〜30日間行なうことができる。培養の過程において、培地は適宜交換することができる。 The mature mast cell of the present invention can be prepared by stimulating mast cells with Wnt. Specifically, mature mast cells can be prepared by culturing the mast cells described above in a medium containing Wnt. More specifically, it can be appropriately adjusted according to the type of mast cell, but it is 1 to 10 5 cells / ml for a medium containing 5 to 100 ng / ml, preferably 20 to 50 ng / ml Wnt. This is achieved by culturing cells of ml to 5 × 10 5 cells / ml in a culture vessel. Mast cell culture can be performed at 37 ° C. for 10 to 30 days. In the course of culturing, the medium can be changed as appropriate.
また本発明においては、ヒト多能性幹細胞由来のマスト細胞を、線維芽細胞と共培養することにより、成熟マスト細胞を作製する。本発明において用いられる線維芽細胞は、ヒト、マウス、ラット、サル等のいかなる生物種であってもよい。例えば、Swiss 3T3線維芽細胞、間葉系幹細胞であるC3H10T1/2細胞、マウスプライマリー皮膚線維芽細胞（MDF）などが挙げられ、好ましくはC3H10T1/2細胞、MDFを用いることができる。培養時のマスト細胞と、線維芽細胞の濃度は、当業者が適宜調節可能であるが、例えばマスト細胞は1×105〜5×105 cells/mlであり、線維芽細胞は0.5×105〜5×105 cells/mlである。ヒト多能性幹細胞由来のマスト細胞と線維芽細胞との細胞数の割合は、マスト細胞：線維芽細胞＝１：0.5〜１の範囲である。 In the present invention, mature mast cells are prepared by co-culturing mast cells derived from human pluripotent stem cells with fibroblasts. The fibroblast used in the present invention may be any biological species such as human, mouse, rat, monkey and the like. Examples thereof include Swiss 3T3 fibroblasts, C3H10T1 / 2 cells that are mesenchymal stem cells, mouse primary skin fibroblasts (MDF), and preferably C3H10T1 / 2 cells and MDF can be used. The concentration of mast cells and fibroblasts during culture can be adjusted as appropriate by those skilled in the art. For example, mast cells are 1 × 10 5 to 5 × 10 5 cells / ml, and fibroblasts are 0.5 × 10 5. 5 to 5 × 10 5 cells / ml. The ratio of the number of mast cells derived from human pluripotent stem cells and fibroblasts is in the range of mast cells: fibroblasts = 1: 0.5-1.
上記、マスト細胞を培養する際に使用する培地は、自体公知の基本培地に抗生物質（例えば 100 IU/mlペニシリン、100 mg/mlストレプトマイシン）や血清又はアルブミン（例えば、牛胎児血清、ウシアルブミン、ヒトアルブミン等）を10〜20%程度添加したものを使用することができる。基本培地としては、RPMI164培地やDMEM培地を使用することができる。また、培地には、適宜SCFやIL-3、IL-6を加え、分化誘導を促進することができる。その他、L-グルタミン、NEAA、ヌクレオシド、β-メルカプトエタノールやその他の成分も必要に応じて適宜添加することができる。
Wntでマスト細胞を刺激して成熟マスト細胞を作製する場合は、基本培地にSCFとIL-6とを添加した培地を用いて共培養を行うことが好ましい。より具体的には、50〜100ng/mlのSCF、50〜100ng/mlのIL-6を含む培地を用いてマスト細胞を培養容器にて培養し、Wntでマスト細胞を刺激することが好適である。
マスト細胞と線維芽細胞とを共培養して成熟マスト細胞を作製する場合は、基本培地にSCFとIL-6を添加した培地を用いて共培養を行うことが好ましい。より具体的には、50〜100ng/mlのSCFと50〜100ng/mlのIL-6を含む培地に対して、マスト細胞と線維芽細胞を培養容器にて培養することが好適である。
The medium used for culturing mast cells is a basic medium known per se, such as antibiotics (eg, 100 IU / ml penicillin, 100 mg / ml streptomycin), serum or albumin (eg, fetal bovine serum, bovine albumin, What added about 10 to 20% of human albumin etc. can be used. As the basic medium, RPMI164 medium or DMEM medium can be used. Moreover, differentiation induction can be promoted by appropriately adding SCF, IL-3, or IL-6 to the medium. In addition, L-glutamine, NEAA, nucleoside, β-mercaptoethanol and other components can be added as necessary.
When producing mast cells by stimulating mast cells with Wnt, it is preferable to perform co-culture using a medium in which SCF and IL-6 are added to the basic medium. More specifically, it is preferable to culture mast cells in a culture vessel using a medium containing 50 to 100 ng / ml SCF and 50 to 100 ng / ml IL-6, and stimulate the mast cells with Wnt. is there.
When mast cells and fibroblasts are co-cultured to produce mature mast cells, it is preferable to perform co-culture using a medium in which SCF and IL-6 are added to the basic medium. More specifically, it is preferable to culture mast cells and fibroblasts in a culture vessel with respect to a medium containing 50 to 100 ng / ml SCF and 50 to 100 ng / ml IL-6.
上記により得られた培養細胞が成熟マスト細胞であるかは、FcεRIやc-kitの発現を確認するほか、プロテアーゼ活性（Tryptase活性、CPA活性）、Tryptaseやキマーゼ（Chymase）、CD81、HDCの発現、サフラニン染色陽性度等によって確認することができる。 In addition to confirming the expression of FcεRI and c-kit, whether the cultured cells obtained above are mature mast cells, as well as expression of protease activity (Tryptase activity, CPA activity), Tryptase, Chymase, CD81, and HDC It can be confirmed by the degree of positive safranin staining.
本発明は上記方法により作製された「成熟マスト細胞」にも及ぶ。 The present invention also extends to “mature mast cells” produced by the above method.
以下、本発明の理解を深めるために本発明をなすに至った経緯を参考例に示し、具体的な発明の内容を実施例及び実験例を示して説明するが、これらは本発明の範囲を限定するものではないことはいうまでもない。 Hereinafter, in order to deepen the understanding of the present invention, the background that led to the present invention will be shown in a reference example, and the specific contents of the invention will be described with reference to examples and experimental examples. Needless to say, this is not a limitation.
（参考例１）マウス骨髄由来マスト細胞（BMMC）の作製
本参考例では、BMMCの作製について、説明する。
C57BL/6マウスから骨髄を採取し、10 ng/mlのIL-3を含むRPMI1640培地（100 IU/ml ペニシリン、100 mg /mlストレプトマイシン、10%牛胎児血清含有）に骨髄細胞が5×105 cells/mlとなるように培養容器に播種し、37℃で培養した。5日に1度培地を交換した。4週間培養後には99%以上の細胞がFcεRI陽性c-kit陽性のマスト細胞で占められるようになった。回収した細胞をBMMCとして以下の実施例で使用した 。
Reference Example 1 Preparation of Mouse Bone Marrow-Derived Mast Cell (BMMC) In this reference example, preparation of BMMC will be described.
Bone marrow was collected from C57BL / 6 mice, and 5 × 10 5 bone marrow cells in RPMI1640 medium (100 IU / ml penicillin, 100 mg / ml streptomycin, containing 10% fetal bovine serum) containing 10 ng / ml IL-3 It seed | inoculated to the culture container so that it might become cells / ml, and it culture | cultivated at 37 degreeC. The medium was changed once every 5 days. After culturing for 4 weeks, more than 99% of cells became occupied by FcεRI positive c-kit positive mast cells. The collected cells were used as BMMC in the following examples.
（参考例２）マウスiPS細胞由来マスト細胞（iPSMC）の作製
本参考例では、iPSMCの作製について、説明する。
マウスiPS細胞（細胞の種類; 38C2、京都大学、山中伸弥教授から供与）は、EB培地（DMEM培地、ペニシリン、ストレプトマイシン、15%牛胎児血清、2 mM L-グルタミン、NEAA（非必須アミノ酸、Invitrogen）、ヌクレオシド（Millipore）、100μM β-メルカプトエタノール）に1×103 cells/100μl/wellとなるように低接着細胞培養容器（Lipidure(R)コート96well プレート：Nunc）に播種した。2日後にEB培地を80μl/wellを添加し、5日後に培地交換を行い、7日目にEB（胚様体）を回収した。その後、分化用培地１（DMEM培地、ペニシリン、ストレプトマイシン、15%牛胎児血清、2 mM L-グルタミン、NEAA、ヌクレオシド、100μM β-メルカプトエタノール、30 ng/ml IL-6、30 ng/ml IL-3、100 ng/ml SCF）に懸濁後、ペトリ皿に播種した。さらに7日後、細胞を回収し、分化用培地２（DMEM培地、ペニシリン、ストレプトマイシン、15%牛胎児血清、2 mM L-グルタミン、NEAA、ヌクレオシド、100μM β-メルカプトエタノール、30 ng/ml IL-3、100 ng/ml SCF）に懸濁後、培養皿に播種し、7日間培養を行い、得られた細胞をiPSMCとして以下の実施例で使用した。
Reference Example 2 Production of Mouse iPS Cell-Derived Mast Cell (iPSMC) In this reference example, the production of iPSMC will be described.
Mouse iPS cells (cell type; 38C2, provided by Kyoto University, Professor Shinya Yamanaka), EB medium (DMEM medium, penicillin, streptomycin, 15% fetal calf serum, 2 mM L-glutamine, NEAA (non-essential amino acid, Invitrogen ), Nucleoside (Millipore), 100 μM β-mercaptoethanol) was seeded in a low adhesion cell culture vessel (Lipidure (R) -coated 96-well plate: Nunc) at 1 × 10 3 cells / 100 μl / well. Two days later, 80 μl / well of EB medium was added, medium was changed after five days, and EB (embryoid body) was collected on the seventh day. Thereafter, differentiation medium 1 (DMEM medium, penicillin, streptomycin, 15% fetal calf serum, 2 mM L-glutamine, NEAA, nucleoside, 100 μM β-mercaptoethanol, 30 ng / ml IL-6, 30 ng / ml IL- (3, 100 ng / ml SCF) and seeded on a Petri dish. After another 7 days, the cells were collected, and differentiation medium 2 (DMEM medium, penicillin, streptomycin, 15% fetal calf serum, 2 mM L-glutamine, NEAA, nucleoside, 100 μM β-mercaptoethanol, 30 ng / ml IL-3 , 100 ng / ml SCF), seeded on a culture dish, cultured for 7 days, and the obtained cells were used as iPSMC in the following examples.
（参考例３）従来法によるマスト細胞の成熟化の確認
本参考例では、マスト細胞をSwiss 3T3細胞株又はOP9細胞株と共培養することによる成熟マスト細胞の作製について説明する。
参考例１で作製したBMMCについて、100 ng/mlのSCFを含むRPMI1640培地に5×105 cells/mlとなるように播種し、マイトマイシンCで処理したSwiss 3T3細胞株又はOP9細胞株と共に計16日間培養した。なお、4日に1度、新しく播種したSwiss 3T3細胞株又はOP9細胞株上に播種しなおした。成熟化は、サフラニンOによるプロテオグリカンの染色及びアルシアンブルーによるグリコサミノグリカンの染色、並びにプロテアーゼ活性（Tryptase活性、CPA活性）の測定により行った。Tryptaseは、マスト細胞顆粒内に豊富に含まれる中性セリンプロテアーゼで、アルギニン、リジン残基でタンパク質を切断する酵素であり、Tryptase活性は合成基質（S-2288、積水メディカル）を用いて測定した。また、CPAは基質の C-末端のペプチド結合を加水分解する酵素であり、CPA活性は合成基質（M-2245、Bachem）を用いて測定した。
(Reference Example 3) Confirmation of mast cell maturation by conventional method This reference example describes the production of mature mast cells by co-culturing mast cells with Swiss 3T3 cell line or OP9 cell line.
The BMMC prepared in Reference Example 1 was seeded in RPMI1640 medium containing 100 ng / ml SCF at 5 × 10 5 cells / ml and treated with mitomycin C together with the Swiss 3T3 cell line or OP9 cell line. Cultured for days. In addition, once every 4 days, the cells were seeded again on the newly seeded Swiss 3T3 cell line or OP9 cell line. Maturation was performed by staining proteoglycan with safranin O, staining with glycosaminoglycan with alcian blue, and measuring protease activity (Tryptase activity, CPA activity). Tryptase is a neutral serine protease that is abundant in mast cell granules. It is an enzyme that cleaves proteins with arginine and lysine residues. Tryptase activity was measured using a synthetic substrate (S-2288, Sekisui Medical). . CPA is an enzyme that hydrolyzes the peptide bond at the C-terminal of the substrate, and CPA activity was measured using a synthetic substrate (M-2245, Bachem).
上記の結果、培養後得られた細胞について、Swiss 3T3細胞株又はOP9細胞株で培養したBMMCはサフラニンOによる染色が認められプロテアーゼ活性も共培養しないBMMCに比べて高い活性が認められた。その結果、いずれの場合もマスト細胞の成熟化が観察された（図１参照）。 As a result of the above, BMMC cultured in Swiss 3T3 cell line or OP9 cell line was found to be stained with safranin O, and the activity obtained after culture was higher than that of BMMC not co-cultured with protease. As a result, maturation of mast cells was observed in all cases (see FIG. 1).
（参考例４）マスト細胞表面マーカーの確認
本参考例では、１．未熟マスト細胞（BMMC）、２．成熟マスト細胞（BMMC+SCF+IL-3）について、細胞表面マーカーを確認した。各マスト細胞は、以下の方法で作製した。
１．BMMC：参考例１で作製したものを用いた。
２．BMMC+SCF+IL-3：参考例１で作製したBMMC 5×105 cells/mlについて、100 ng/mlのSCF及び1 ng/mlのIL-3を含むRPMI1640培地で20日間培養し、浮遊細胞を回収した。
(Reference Example 4) Confirmation of mast cell surface marker In this reference example, 1. immature mast cells (BMMC); Cell surface markers were confirmed for mature mast cells (BMMC + SCF + IL-3). Each mast cell was prepared by the following method.
1. BMMC: The one produced in Reference Example 1 was used.
2. BMMC + SCF + IL-3: BMMC 5 × 10 5 cells / ml prepared in Reference Example 1 were cultured in RPMI1640 medium containing 100 ng / ml SCF and 1 ng / ml IL-3 for 20 days and suspended Cells were collected.
その結果、各種マスト細胞にはWnt受容体が発現していることが確認された（図２参照）。 As a result, it was confirmed that Wnt receptor was expressed in various mast cells (see FIG. 2).
（参考例５）液性因子の探索
参考例３において、マスト細胞であるBMMCをOP9細胞株又はSwiss 3T3細胞株と共培養したことで、BMMCが成熟マスト細胞の性質を示したことが確認されたことから、OP9細胞株及びSwiss 3T3細胞株が産生する液性因子を確認した（図３参照）。その結果、OP9細胞株及びSwiss 3T3細胞株に共通して発現される液性因子として、Wnt3、Wnt4、Wnt5a等の種々のWntの発現が確認された（図３参照）。
(Reference Example 5) Search for humoral factors In Reference Example 3, it was confirmed that BMMC, which is a mast cell, was co-cultured with OP9 cell line or Swiss 3T3 cell line, and that BMMC exhibited the properties of mature mast cells. Thus, humoral factors produced by the OP9 cell line and the Swiss 3T3 cell line were confirmed (see FIG. 3). As a result, the expression of various Wnts such as Wnt3, Wnt4, Wnt5a, etc. was confirmed as humoral factors that are commonly expressed in the OP9 cell line and the Swiss 3T3 cell line (see FIG. 3).
（実施例１）Wnt経路活性化によるマスト細胞の成熟化について
本実施例では、参考例５の結果に基づいて、Wnt経路の活性化がマスト細胞の成熟化に関与するのではないかと考えた。そこで、GSK3β阻害剤である6-Bromoindirubin-3'-oxime（BIO）を用いたWntシグナルの活性化と、マスト細胞の成熟化について検討を行った。
(Example 1) Mast cell maturation by Wnt pathway activation In this example, based on the results of Reference Example 5, it was considered that Wnt pathway activation might be involved in mast cell maturation. . Therefore, we investigated Wnt signal activation and mast cell maturation using 6-Bromoindirubin-3'-oxime (BIO), a GSK3β inhibitor.
BIOによって、GSK3β阻害と細胞質β-cateninの安定化を伴い、Wntシグナルの活性化が認められる。そこで、参考例１で作製したBMMCについて、BIO（0.5μM、1.0μM）を含む系と、コントロールとしてBIOの代わりにDMSOを含む系で培養したときのマスト細胞の成熟化について確認した。BMMC 5×105 cells/mlを、BIO又はDMSOの他、100 ng/mlのSCF及び 1 ng/mlのIL-3を含むRPMI1640培地で20日間培養し、5日に1度培地を交換した（図４Ａ参照）。成熟化はCD81の発現、プロテアーゼ活性の測定、及びHDC、MCP5、CPAの発現確認により行った。CD81はFACS分析により測定した。プロテアーゼ活性は参考例３と同手法により測定した。HDCは哺乳類の末梢におけるヒスタミンの合成に関与する蛋白である。MCP5及びCPAは、マスト細胞内に存在する酵素である。HDC、MCP5、CPAの発現は、RT-PCRの手法により確認した。 BIO activates the Wnt signal with inhibition of GSK3β and stabilization of cytoplasmic β-catenin. Therefore, maturation of mast cells when BMMC prepared in Reference Example 1 was cultured in a system containing BIO (0.5 μM, 1.0 μM) and a system containing DMSO instead of BIO as a control was confirmed. BMMC 5 × 10 5 cells / ml was cultured in RPMI1640 medium containing 100 ng / ml SCF and 1 ng / ml IL-3 in addition to BIO or DMSO for 20 days, and the medium was changed once every 5 days. (See FIG. 4A). Maturation was performed by measuring CD81 expression, measuring protease activity, and confirming the expression of HDC, MCP5, and CPA. CD81 was measured by FACS analysis. Protease activity was measured by the same method as in Reference Example 3. HDC is a protein involved in the synthesis of histamine in the periphery of mammals. MCP5 and CPA are enzymes present in mast cells. The expression of HDC, MCP5, and CPA was confirmed by RT-PCR.
上記の結果、BIOを含む系で培養した場合に、コントロールに比べて細胞表面にCD81がやや強く発現していることが確認された（図４Ｂ参照）。また、Tryptase活性やCPA活性もBIOの濃度依存的に上昇していることが確認された（図４Ｃ参照）。さらに、HDC、MCP5、CPA mRNAの発現に関しても、BIOの濃度依存的に上昇していることが確認された（図４Ｄ参照）。このことから、Wntシグナルの活性化により、マスト細胞が成熟化することが示唆された。 As a result, when cultured in a system containing BIO, it was confirmed that CD81 was expressed more strongly on the cell surface than in the control (see FIG. 4B). Moreover, it was confirmed that Tryptase activity and CPA activity also increased in a BIO concentration-dependent manner (see FIG. 4C). Furthermore, the expression of HDC, MCP5, and CPA mRNA was also confirmed to increase in a BIO concentration-dependent manner (see FIG. 4D). This suggested that mast cells mature by activation of the Wnt signal.
（実施例２）Wnt5aによるマスト細胞の成熟化について
本実施例では、BMMCについて、Wnt5a（25 ng/ml）を含む系で培養したときのマスト細胞の成熟化について確認した。
(Example 2) Mast cell maturation by Wnt5a In this example, maturation of mast cells when BMMC was cultured in a system containing Wnt5a (25 ng / ml) was confirmed.
参考例１で作製したBMMC 5×105 cells/mlを、Wnt5aの他、100ng/mlのSCF及び1 ng/mlのIL-3を含むRPMI1640培地で20日間培養した。5日に1度培地を交換した（図５Ａ参照）。成熟化は、CD81の発現、Tryptase活性及びCPA活性の測定、並びにHDCの発現確認により行った。Tryptase活性及びCPA活性は参考例３と同手法により測定した。CD81、HDC、MCP5の発現は、実施例１と同手法により確認した。 BMMC 5 × 10 5 cells / ml prepared in Reference Example 1 were cultured for 20 days in RPMI1640 medium containing 100 ng / ml SCF and 1 ng / ml IL-3 in addition to Wnt5a. The medium was changed once every 5 days (see FIG. 5A). Maturation was performed by measuring CD81 expression, Tryptase activity and CPA activity, and confirming HDC expression. Tryptase activity and CPA activity were measured by the same method as in Reference Example 3. The expression of CD81, HDC and MCP5 was confirmed by the same method as in Example 1.
上記の結果、Wnt5aを含む系で培養した場合に、コントロールに比べて細胞表面にCD81がやや強く発現した（図５Ｂ参照）。また、プロテアーゼ活性も上昇し、さらに、HDC及びMCP5 mRNAの発現も有意に高い値を示した（図５Ｃ、Ｄ参照）。このことから、Wnt5aによるWntシグナルの活性化により、マスト細胞が成熟化することが確認された。 As a result, when cultured in a system containing Wnt5a, CD81 was expressed slightly more strongly on the cell surface than in the control (see FIG. 5B). Protease activity also increased, and HDC and MCP5 mRNA expression also showed significantly high values (see FIGS. 5C and D). From this, it was confirmed that mast cells mature by activation of Wnt signal by Wnt5a.
（実施例３）Wnt5aによるマスト細胞の成熟化について
参考例２で作製したiPS細胞由来マスト細胞について、Wnt5aを含む系で培養したときのマスト細胞の成熟化について確認した。EBを、100 ng/mlのSCF、30 ng/mlのIL-3及び、30 ng/mlのIL-6を含む分化用培地１で7日間培養したのち、100 ng/mlのSCF及び30 ng/mlのIL-3を含む分化用培地２で7日間培養した。さらに25 ng/mlのWnt5aの他、100 ng/mlのSCF及び30 ng/mlのIL-3を含む分化用培地２で14日間培養した（図６Ａ参照）。成熟化は、Tryptase活性及びCPA活性の測定、並びにHDCの発現確認により行った。Tryptase活性は参考例３と同手法により、CPA活性及びHDCの発現は実施例２と同手法により測定した。
(Example 3) Mast cell maturation by Wnt5a The iPS cell-derived mast cells prepared in Reference Example 2 were confirmed for mast cell maturation when cultured in a system containing Wnt5a. EB was cultured for 7 days in differentiation medium 1 containing 100 ng / ml SCF, 30 ng / ml IL-3 and 30 ng / ml IL-6, then 100 ng / ml SCF and 30 ng The cells were cultured in differentiation medium 2 containing / ml IL-3 for 7 days. Furthermore, in addition to 25 ng / ml Wnt5a, the cells were cultured for 14 days in differentiation medium 2 containing 100 ng / ml SCF and 30 ng / ml IL-3 (see FIG. 6A). Maturation was performed by measuring Tryptase activity and CPA activity, and confirming the expression of HDC. Tryptase activity was measured by the same method as in Reference Example 3, and CPA activity and HDC expression were measured by the same method as in Example 2.
上記の結果、Wnt5aを含む系で培養した場合に、コントロールに比べてTryptase活性及びCPA活性が上昇していることが確認された（図６Ｂ参照）。さらにHDCの発現も有意に高い値を示した（図６Ｃ参照）。このことから、Wnt5aによるWntシグナルの活性化により、マスト細胞が成熟化することが確認された。 As a result, it was confirmed that when cultured in a system containing Wnt5a, Tryptase activity and CPA activity were increased compared to the control (see FIG. 6B). Furthermore, the expression of HDC also showed a significantly high value (see FIG. 6C). From this, it was confirmed that mast cells mature by activation of Wnt signal by Wnt5a.
（実験例１）Wnt5aによるマスト細胞の成熟化の確認試験
本実験例では、参考例１で作製したBMMCについて、Swiss 3T3細胞株と共に抗Wnt5a抗体を含む系で培養したときのマスト細胞の成熟化について確認した。5×105 cells/mlのBMMC、Swiss 3T3細胞株、及び1μg/mlの抗Wnt5a抗体の他、100 ng/mlのSCFを含むRPMI1640培地で16日間培養した（図７Ａ参照）。抗Wnt5a抗体に対するコントロールとしてnrIgG抗体を用いた。成熟化は、CD81の発現及びTryptase活性の測定により行った。測定は、前述の参考例及び実施例と同手法により行った。
(Experimental example 1) Confirmation test of maturation of mast cells by Wnt5a In this experimental example, maturation of mast cells when BMMC prepared in Reference Example 1 was cultured in a system containing anti-Wnt5a antibody together with the Swiss 3T3 cell line. Confirmed about. In addition to 5 × 10 5 cells / ml BMMC, Swiss 3T3 cell line, and 1 μg / ml anti-Wnt5a antibody, the cells were cultured for 16 days in RPMI1640 medium containing 100 ng / ml SCF (see FIG. 7A). NrIgG antibody was used as a control for anti-Wnt5a antibody. Maturation was performed by measuring CD81 expression and tryptase activity. The measurement was performed by the same method as in the reference examples and examples described above.
上記の結果、抗Wnt5a抗体を含む系で培養した場合に、コントロールに比べて細胞表面にCD81の発現がやや抑制されたことが確認され、Tryptase活性も抑制していることが確認された（図７Ｂ，Ｃ参照）。このことからも、Wnt5aによるWntシグナルの活性化により、マスト細胞が成熟化することが確認された。 As a result, when cultured in a system containing an anti-Wnt5a antibody, it was confirmed that the expression of CD81 was slightly suppressed on the cell surface compared to the control, and that Tryptase activity was also suppressed (Fig. 7B, C). This also confirmed that mast cells matured by Wnt5a activation of Wnt5a.
（参考例６）マウスiPS細胞由来マスト細胞の作製
（１）マウスiPS細胞から血液前駆細胞への分化誘導
分化誘導開始の前日に50 Gy放射線照射し増殖を止めたC3H10T1/2細胞株をゼラチンコートした10 cm培養皿に7 x 105個で播種し、フィーダー細胞として用いた。マウスiPS細胞（細胞の種類; 38C2、京都大学、山中伸弥教授から供与）は、5〜10×104個/10 cm 培養皿となるようにフィーダー細胞上に播種した。細胞の播き直しをは行わず、50 ng/ml vascular endothelial growth factor（VEGF）（Peprotech社）含有培地を9日目までは3日毎、9日目から15日目までは2日毎に交換することにより血液前駆細胞の誘導を行った（Takayama N, Nishikii H, Usui J, Tsukui H, Sawaguchi A, Hiroyama T, Eto K, Nakauchi H. Blood. 2008 Jun 1;111(11):5298-306. doi: 10.1182/blood-2007-10-117622.）。セルソーターにてCD34+CD43+血液前駆細胞を分離した。
(Reference Example 6) Preparation of mouse iPS cell-derived mast cells (1) Induction of differentiation from mouse iPS cells to blood progenitor cells Gelatin-coated C3H10T1 / 2 cell line that was irradiated with 50 Gy the day before the start of differentiation induction and stopped growing 7 × 10 5 cells were inoculated into 10 cm culture dishes and used as feeder cells. Mouse iPS cells (cell type; 38C2, provided by Kyoto University, Professor Shinya Yamanaka) were seeded on feeder cells so as to form 5-10 × 10 4 cells / 10 cm culture dish. Change the medium containing 50 ng / ml vascular endothelial growth factor (VEGF) (Peprotech) every 3 days from day 9 and every 2 days from day 9 to day 15 without reseeding the cells. Blood progenitor cells were induced by (Takayama N, Nishikii H, Usui J, Tsukui H, Sawaguchi A, Hiroyama T, Eto K, Nakauchi H. Blood. 2008 Jun 1; 111 (11): 5298-306. Doi : 10.1182 / blood-2007-10-117622.). CD34 + CD43 + blood progenitor cells were separated using a cell sorter.
（２）マウスiPS細胞由来血液前駆細胞からマスト細胞への分化誘導
上述の（１）にて得られたCD34+CD43+血液前駆細胞を、200 ng/ml SCF、50 ng/ml IL-6、5 ng/ml IL-3含有培地の懸濁後、MethoCult H4236メチルセルロース培地（Stem Cell）と混和し、low binding 24 well プレート（Nunc）に播種した。2週間後、200 ng/ml SCFおよび50 ng/ml IL-6含有IMDM（培地）とMethoCult H4236メチルセルロース培地を混和し重層した。その2週間後（Week 5）、200 ng/ml SCFおよび50 ng/ml IL-6含有IMDMをさらに重層した（Saito H, Kato A, Matsumoto K, Okayama Y. Nat Protoc. 2006;1(4):2178-83.）。メチルセルロース中での培養6週間目に、顕微鏡下でコロニーをピックアップし、さらに100 ng/mL SCFおよび100 ng/mL IL-6含有培地にて２週間培養し、得られた細胞を、マウスiPS細胞由来のマスト細胞として、以下の実施例で使用した。
(2) Induction of differentiation from mouse iPS cell-derived blood progenitor cells to mast cells The CD34 + CD43 + blood progenitor cells obtained in (1) above were treated with 200 ng / ml SCF, 50 ng / ml IL-6, After suspending the medium containing 5 ng / ml IL-3, it was mixed with MethoCult H4236 methylcellulose medium (Stem Cell) and seeded on a low binding 24 well plate (Nunc). Two weeks later, IMDM (medium) containing 200 ng / ml SCF and 50 ng / ml IL-6 and MethoCult H4236 methylcellulose medium were mixed and overlaid. Two weeks later (Week 5), IMDM containing 200 ng / ml SCF and 50 ng / ml IL-6 was further overlaid (Saito H, Kato A, Matsumoto K, Okayama Y. Nat Protoc. 2006; 1 (4) : 2178-83.). At 6 weeks of culturing in methylcellulose, colonies were picked up under a microscope and further cultured in a medium containing 100 ng / mL SCF and 100 ng / mL IL-6 for 2 weeks. The obtained cells were used as mouse iPS cells. The derived mast cells were used in the following examples.
（実施例４）線維芽細胞との共培養法によるマスト細胞の成熟化
参考例６の方法により得られた、マウスiPS細胞由来のマスト細胞（1×105cells/ml）を100 ng/mL SCF、100 ng/mL IL-6存在下で、前日播種した種々の支持細胞（ヒト臍帯静脈血管内皮細胞（HUVEC）（1×105cells/ml）、Swiss 3T3線維芽細胞株（1×105cells/ml）、間葉系幹細胞株であるC3H10T1/2細胞（1×105cells/ml）、マウスプライマリー皮膚線維芽細胞（MDF）（1×105cells/ml））と7日間共培養した。その後、NucleoSpin RNA XS（TaKaRa）を用いてTotal RNAを抽出した。各Total RNAをRNase-free DNase I（New England Biolabs）で処理した後、Superscript VILO cDNA synthesis kit（Life Technologies）を用いて逆転写反応を行い、cDNAを合成した。その後、cDNAをFast SYBR Green Master Mix（Life Technologies）にて増幅し定量した（StepOne Plus ; Life Technologies）。
Example 4 Mast Cell Maturation by Co-culture with Fibroblasts 100 ng / mL of mouse iPS cell-derived mast cells (1 × 10 5 cells / ml) obtained by the method of Reference Example 6 Various supporting cells (human umbilical vein endothelial cells (HUVEC) (1 × 10 5 cells / ml), Swiss 3T3 fibroblast cell line (1 × 10 5 ) seeded the day before in the presence of SCF, 100 ng / mL IL-6 5 cells / ml), mesenchymal stem cell line C3H10T1 / 2 cells (1 × 10 5 cells / ml), mouse primary skin fibroblasts (MDF) (1 × 10 5 cells / ml)) for 7 days Cultured. Thereafter, Total RNA was extracted using NucleoSpin RNA XS (TaKaRa). Each total RNA was treated with RNase-free DNase I (New England Biolabs), and then reverse transcription was performed using Superscript VILO cDNA synthesis kit (Life Technologies) to synthesize cDNA. Then, cDNA was amplified and quantified by Fast SYBR Green Master Mix (Life Technologies) (StepOne Plus; Life Technologies).
本実施例では、生体内でマスト細胞が血管周囲に多く存在することからヒト臍帯静脈血管内皮細胞（HUVEC）、Swiss 3T3線維芽細胞株、間葉系幹細胞株であるC3H10T1/2細胞、マウスプライマリー皮膚線維芽細胞（MDF）との共培養を試みた。その結果、マウスiPS細胞由来マスト細胞は、C3H10T1/2細胞株やMDFと共培養することによりキマーゼやHDCm RNA量の増加が観察された。よって、線維芽細胞と共培養することでマウスマスト細胞の成熟化も促進されることが示された（図８）。
In this example, since there are many mast cells around the blood vessel in vivo, human umbilical vein vascular endothelial cells (HUVEC), Swiss 3T3 fibroblast cell line, C3H10T1 / 2 cell which is a mesenchymal stem cell line, mouse primary Co-culture with dermal fibroblasts (MDF) was attempted. As a result, mast cells derived from mouse iPS cells were observed to increase chymase and HDC mRNA levels when cocultured with C3H10T1 / 2 cell line and MDF. Therefore, it was shown that the maturation of mouse mast cells is promoted by co-culture with fibroblasts (FIG. 8).
（実施例５）Wnt5aを用いたマスト細胞の成熟化
参考例６の方法で得られた、マウスiPS細胞由来のマスト細胞（1×105cells/ml）を100 ng/mL SCF、100 ng/mL IL-6、50 ng/mL Wnt5a存在下で2週間培養した。得られた細胞から、上述した方法によりcDNAを合成後、Fast SYBR Green Master Mixにて増幅し定量した。
(Example 5) Maturation of mast cells using Wnt5a Mast cells derived from mouse iPS cells (1 × 10 5 cells / ml) obtained by the method of Reference Example 6 were treated with 100 ng / mL SCF, 100 ng / The cells were cultured for 2 weeks in the presence of mL IL-6 and 50 ng / mL Wnt5a. CDNA was synthesized from the obtained cells by the method described above, and then amplified and quantified with Fast SYBR Green Master Mix.
本実施例では、マウスiPS細胞由来マスト細胞の成熟化を、Wnt5aが促進するか否かについて検討を行った。マウスiPS細胞由来マスト細胞にWnt5aを作用させた結果、HDC mRNA量の増加が観察された（図９）。 In this example, it was examined whether Wnt5a promotes maturation of mouse iPS cell-derived mast cells. As a result of Wnt5a acting on mouse iPS cell-derived mast cells, an increase in the amount of HDC mRNA was observed (FIG. 9).
（参考例７）ヒトiPS細胞から血液前駆細胞への分化誘導（EB形成法）
参考例６の（１）の代替法として、以下のEB形成法により、ヒトiPS細胞から血液前駆細胞への分化誘導を行った。
ヒトiPS細胞（細胞の種類; 201B7、京都大学、山中伸弥教授から供与）をAccutase（Millipore）により培養ディッシュから剥離し、10 μM Y27632（Wako）を含むEB形成培地［50 μg/ml アスコルビン酸（Sigma）と450 μM Monothioglycerol（Sigma）、付属のサプリメントを加えたmTeSR（Stem Cell）］中でピペッティングすることにより単細胞に解離した。その後、1×106個のiPS細胞と前日放射線処理した6×105個C3H10T1/2細胞を2 ng/ml ActivinA（R&D Systeme）、2 ng/ml bone morphogenic protein 4（BMP4）（R&D System）、10 μM Rho-associated coiled-coil forming kinase （ROCK）inhibitor （Y-27632 ; Wako）を含むEB形成培地に懸濁し、ペトリディッシュに播種した。2日後（Day 2）、2 ng/ml BMP4、5 ng/ml VEGFを含むEB分化培地［50 μg/ml アスコルビン酸（Sigma）と450 μM Monothioglycerol（Sigma社）、2 mM L-グルタミン（Life Technologies）、インスリントランスフェリン（Life Technologies）を加えたIMDM（Sigma）］に置き換えた。その2日後（Day 4）、2 ng/ml BMP4、5 ng/ml VEGF、10 μM SB431542（Wako）を含むEB分化培地で半分培地を交換し、更に2日培養した（SB431542の終濃度は5 μM）。分化誘導から6日目（Day 6）に、2 ng/ml BMP4、5 ng/ml VEGF、20 ng/ml stem cell factor （SCF; Pepotech）、20 ng/ml Thrombopoietin（TPO; Peprotech）を含むEB分化培地に培地を交換した。2日後（Day 8）に半分培地交換を行い、血液前駆細胞の誘導を行った。得られた血液前駆細胞は、参考例６（２）の方法等により、マスト細胞の分化誘導に用いることが可能である。
(Reference Example 7) Differentiation induction from human iPS cells to blood precursor cells (EB formation method)
As an alternative to Reference Example 6 (1), differentiation induction from human iPS cells to blood precursor cells was performed by the following EB formation method.
Human iPS cells (cell type; 201B7, provided by Kyoto University, Prof. Shinya Yamanaka) were detached from the culture dish with Accutase (Millipore), and EB formation medium containing 10 μM Y27632 (Wako) [50 μg / ml ascorbic acid ( Sigma) and 450 μM Monothioglycerol (Sigma), mTeSR (Stem Cell) supplemented with supplements], and then dissociated into single cells. After that, 1 x 10 6 iPS cells and 6 x 10 5 C3H10T1 / 2 cells treated with the previous day were treated with 2 ng / ml ActivinA (R & D Systeme), 2 ng / ml bone morphogenic protein 4 (BMP4) (R & D System) The suspension was suspended in an EB-forming medium containing 10 μM Rho-associated coiled-coil forming kinase (ROCK) inhibitor (Y-27632; Wako) and seeded in a Petri dish. 2 days later (Day 2), EB differentiation medium containing 2 ng / ml BMP4 and 5 ng / ml VEGF [50 μg / ml ascorbic acid (Sigma) and 450 μM Monothioglycerol (Sigma), 2 mM L-glutamine (Life Technologies) ), IMDM (Sigma) plus insulin transferrin (Life Technologies)]. Two days later (Day 4), half the medium was replaced with EB differentiation medium containing 2 ng / ml BMP4, 5 ng / ml VEGF, and 10 μM SB431542 (Wako), and further cultured for 2 days (the final concentration of SB431542 was 5 μM). EB containing 2 ng / ml BMP4, 5 ng / ml VEGF, 20 ng / ml stem cell factor (SCF; Pepotech), 20 ng / ml Thrombopoietin (TPO; Peprotech) on day 6 after differentiation induction The medium was changed to the differentiation medium. Two days later (Day 8), half of the medium was changed to induce blood precursor cells. The obtained blood progenitor cells can be used for induction of mast cell differentiation by the method of Reference Example 6 (2).
以上詳述したように本発明の方法で作製された成熟マスト細胞によれば、生体内のマスト細胞の機能の理解が一段と深まることが期待される。特にマスト細胞欠損マウス等のモデル動物に移入することで、局所のマスト細胞の機能を解析し、多様な免疫応答に対するマスト細胞による制御機構が明らかにされるものと期待される。さらに、マスト細胞の機能に着目した医薬品の開発に関しても貢献しうるものと期待される。 As described above in detail, according to the mature mast cell produced by the method of the present invention, it is expected that the understanding of the function of the mast cell in the living body will be further deepened. In particular, by transferring to a model animal such as a mast cell-deficient mouse, it is expected that the function of the local mast cell will be analyzed and the control mechanism by the mast cell for various immune responses will be clarified. Furthermore, it is expected to contribute to the development of pharmaceuticals that focus on the function of mast cells.
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