JP2022017977A - Cell, composition and composition for treatment - Google Patents
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Abstract
Description
本発明は、細胞、組成物に関する。
本発明の細胞は、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）であることを特徴とする。本発明の細胞群は、本発明の細胞を４０％以上含む細胞群である。本発明はまた、本発明の細胞または細胞群を含む、虚血性疾患、炎症性疾患又は難治療性創傷の治療用組成物に関する。
The present invention relates to cells and compositions.
The cell of the present invention is characterized by being CD14 (+) CD206 (+) CXCR4 (+). The cell group of the present invention is a cell group containing 40% or more of the cells of the present invention. The present invention also relates to therapeutic compositions for ischemic, inflammatory or refractory wounds comprising the cells or groups of cells of the invention.
近年、虚血性疾患を対象として、骨髄単核球移植治療や末梢血幹細胞採取による血管内皮前駆細胞（Ｅｎｄｏｔｈｅｌｉａｌ ｐｒｏｇｅｎｉｔｏｒ ｃｅｌｌ；ＥＰＣ）を用いた細胞移植治療法がおこなわれるようになった。それゆえ、ＥＰＣを大量に培養する技術が特に求められていた。ＣＤ３４及び／又はＣＤ１３３陽性細胞からの血管内皮前駆細胞の生体外増幅方法は、効率的なＥＰＣの培養技術を提供することを可能にした（特許文献１）。さらに血管内皮細胞分化動態解析方法によって内皮細胞様大コロニー（分化型ＥＰＣコロニー）形成細胞と内皮細胞様小コロニー（未分化型ＥＰＣコロニー）形成細胞が存在することが明らかになり細胞移植による治療効果を予測、把握することを可能にした（特許文献２）。また骨髄単核球からＣＤ３４及び／又はＣＤ１３３陽性細胞を効率的に増幅させる方法を示した（特許文献３）。 In recent years, for ischemic diseases, bone marrow mononuclear cell transplantation therapy and cell transplantation therapy using vascular endothelial progenitor cell (EPC) by collecting peripheral blood stem cells have been performed. Therefore, a technique for culturing a large amount of EPC has been particularly required. The method of in vitro amplification of vascular endothelial progenitor cells from CD34 and / or CD133 positive cells has made it possible to provide an efficient EPC culture technique (Patent Document 1). Furthermore, it was clarified by the vascular endothelial cell differentiation dynamics analysis method that there are endothelial cell-like large colony (differentiated EPC colony) -forming cells and endothelial cell-like small colonies (undifferentiated EPC colony) forming cells, and the therapeutic effect of cell transplantation. (Patent Document 2). Further, a method for efficiently amplifying CD34 and / or CD133-positive cells from bone marrow mononuclear cells has been shown (Patent Document 3).
国際公開ＷＯ２０１４／０５１１５４に示されるように、骨髄、臍帯血又は末梢血由来の単核球画分から血管内皮前駆細胞又は抗炎症・免疫寛容誘導細胞が富化した細胞群を増幅させる方法（以下、本明細書において、「ＱＱ－ＭＮＣ法」）と呼称する場合がある）を記載している。当該方法は、（１）幹細胞因子（Ｓｔｅｍ ｃｅｌｌ ｆａｃｔｏｒ；ＳＣＦ），（２）インターロイキン６（Ｉｎｔｅｒｌｅｕｋｉｎ－６；ＩＬ－６），（３）ＦＭＳ様チロシンキナーゼ３リガンド（ＦＭＳ－ｌｉｋｅ ｔｙｒｏｓｉｎｅ ｋｉｎａｓｅ ３ ｌｉｇａｎｄ；ＦＬ），（４）トロンボポエチン（Ｔｈｒｏｍｂｏｐｏｉｅｔｉｎ；ＴＰＯ）及び（５）血管内皮細胞増殖因子（Ｖａｓｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ；ＶＥＧＦ）の５つの因子を含む無血清培地で、単核球を培養することにより、ＥＰＣを含む細胞群を生体外で増幅する、というものである。
本発明者らは、さらに、特願２０１９－２３１６０６（非公開）に示されるように、鋭意研究に努めた結果、骨髄、臍帯血又は末梢血由来の単核球を、幹細胞因子、インタ一ロイキン６、ＦＭＳ様チロシンキナーゼ３リガンド、トロンボポエチン及び血管内皮細胞増殖因子からなる群から選択される４以下の因子、並びに、血清、を含む培地中で、培養することにより、血管再生能及び創傷治癒能を有し、かつ、国際公開ＷＯ２０１４／０５１１５４に示した細胞群とは異なる新たな細胞群の取得に成功した。
As shown in WO2014 / 051154, a method for amplifying a cell group enriched with vascular endothelial progenitor cells or anti-inflammatory / immunotolerance-inducing cells from a mononuclear cell fraction derived from bone marrow, cord blood or peripheral blood (hereinafter, In the present specification, it may be referred to as "QQ-MNC method")). The methods include (1) stem cell factor (SCF), (2) interleukin-6 (IL-6), and (3) FMS-like tyrosine kinase 3 led. By culturing mononuclear cells in a serum-free medium containing five factors: FL), (4) Thrombopoietin (TPO) and (5) Vascular Endothelial Growth Factor; VEGF. It is to amplify a group of cells including EPC in vitro.
Further, as shown in Japanese Patent Application No. 2019-231606 (unpublished), as a result of diligent research, the present inventors have obtained mononuclear cells derived from bone marrow, cord blood or peripheral blood as a stem cell factor, interleukin. 6. Vascular regeneration ability and wound healing ability by culturing in a medium containing 4 or less factors selected from the group consisting of FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum. And succeeded in obtaining a new cell group different from the cell group shown in the international publication WO2014 / 051154.
本発明者らは、特願２０１９－２３１６０６（非公開）で示す細胞群の研究を進めた結果、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞、及び当該細胞を多く含む細胞群を発見し、当該細胞および当該細胞を多く含む細胞群がさらに優れた血管再生能及び創傷治癒能を有することを見出して本願発明を想到した。 As a result of advancing the study of the cell group shown in Japanese Patent Application No. 2019-231606 (closed), the present inventors have found that the cells are CD14 (+) CD206 (+) CXCR4 (+) and the cell group containing a large amount of the cells. The present invention was conceived by discovering that the cell and the cell group containing a large amount of the cell have further excellent vascular regeneration ability and wound healing ability.
本発明は、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞を提供する。
本発明はまた、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞を４０％以上含む細胞群を提供する。
The present invention provides cells that are CD14 (+) CD206 (+) CXCR4 (+).
The present invention also provides a cell group containing 40% or more of cells which are CD14 (+) CD206 (+) CXCR4 (+).
本発明はまた、本発明の細胞または細胞群を含む、虚血性疾患、炎症性疾患又は難治療性創傷の治療用組成物を提供する。 The present invention also provides a therapeutic composition for an ischemic disease, an inflammatory disease or a refractory wound, comprising the cells or groups of cells of the present invention.
限定されるわけではないが、本発明は以下の態様を含む。
［態様１］
ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞。
［態様２］
態様１の細胞を４０％以上含む細胞群。
［態様３］
ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（－）の細胞が２０％以下である、態様２に記載の細胞群。
［態様４］
態様１に記載の細胞、あるいは、態様２又は３に記載の細胞群を含む、虚血性疾患、炎症性疾患又は難治療性創傷の治療用組成物。
［態様５］
虚血性疾患が、四肢虚血である、態様４に記載の治療用組成物、
［態様６］
虚血性疾患が、潰瘍を伴う四肢虚血である、態様４又は５に記載の治療用組成物。
［態様７］
血管新生及び／又は創傷治癒を促進する、態様４－６のいずれか１項に記載の治療用組成物。
The present invention includes, but is not limited to, the following aspects.
[Aspect 1]
A cell that is CD14 (+) CD206 (+) CXCR4 (+).
[Aspect 2]
A cell group containing 40% or more of the cells of aspect 1.
[Aspect 3]
The cell group according to aspect 2, wherein the cells of CD14 (+) CD206 (+) CXCR4 (−) are 20% or less.
[Aspect 4]
A composition for treating an ischemic disease, an inflammatory disease or a refractory wound, which comprises the cell according to the first aspect or the cell group according to the second or third aspect.
[Aspect 5]
The therapeutic composition according to aspect 4, wherein the ischemic disease is limb ischemia.
[Aspect 6]
The therapeutic composition according to
[Aspect 7]
The therapeutic composition according to any one of aspects 4-6, which promotes angiogenesis and / or wound healing.
１．細胞
本発明は、一態様において、細胞に関する。本発明の細胞は、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）であることを特徴とする。
1. 1. Cells The present invention relates to cells in one aspect. The cell of the present invention is characterized by being CD14 (+) CD206 (+) CXCR4 (+).
ＣＤ１４は、ヒト、マウス等で観察される自然免疫系の構成要素の１つである。ＣＤ１４は共受容体として（ＴＬＲ４又はＭＤ－２と共に）働き、細菌に由来するリポ多糖（ＬＰＳ）を認識する。 CD14 is one of the components of the innate immune system observed in humans, mice and the like. CD14 acts as a co-receptor (along with TLR4 or MD-2) and recognizes bacterial-derived lipopolysaccharide (LPS).
ＣＤ２０６（別名：ＭＭＲ、ＣＬＥＣ１３Ｄ、ＭＲＣ１）は、マルチレクチンレセプタ構造を有するＩ型膜受容体である。ＣＤ２０６は、糖鎖を含むタンパク質のマクロファージによる細胞内取り込み、エンドサイトーシスを媒介する。ＣＤ２０６は、潜在的な病原性ウイルスおよび細菌、真菌の表面上の高マンノース構造に結合することが示されており、それらは貪食により排除される。 CD206 (also known as MMR, CLEC13D, MRC1) is a type I membrane receptor with a multilectin receptor structure. CD206 mediates intracellular uptake and endocytosis of sugar chain-containing proteins by macrophages. CD206 has been shown to bind to high mannose structures on the surface of potential pathogenic viruses and bacteria, fungi, which are eliminated by phagocytosis.
ＣＸＣＲ４（別名：ＣＤ１８４）は、細胞膜を７回貫通するＧタンパク質共役受容体（ＧＰＣＲ）である。ＣＸＣＲ４の生理的リガンドは、ＣＸＣケモカインの１種である、ｓｔｏｒｏｍａｌ ｃｅｌｌ－ｄｅｒｉｖｅｄ ｆａｃｔｏｒ－１（ＳＤＦ－１）である。
ＣＸＣＲ４は生体において比較的広範な分布を示し、免疫系や中枢神経系において、様々なＣＤ４（＋）細胞に発現している。血液中ではＣＤ１８４細胞は、好中球、単球、樹状細胞、ＮＫ細胞、Ｂ細胞、Ｔ細胞そして血小板に強く発現している。
CXCR4 (also known as CD184) is a G protein-coupled receptor (GPCR) that penetrates the cell membrane seven times. The physiological ligand for CXCR4 is a statistical cell-developed factor-1 (SDF-1), a type of CXC chemokine.
CXCR4 has a relatively wide distribution in the living body and is expressed in various CD4 (+) cells in the immune system and the central nervous system. In blood, CD184 cells are strongly expressed on neutrophils, monocytes, dendritic cells, NK cells, B cells, T cells and platelets.
ＣＤ１４、ＣＤ２０６、ＣＸＣＲ４のタンパク質及び遺伝子の配列、構造は公知であり、各々が共陽性であること、即ち、ＣＤ１４（＋）、ＣＤ２０６（＋）、ＣＸＣＲ４（＋）は公知方法によって調べることができる。非限定的に、ＣＤ１４、ＣＤ２０６、ＣＸＣＲ４のタンパク質の存在は、例えば、これらのタンパク質に特異的に結合する物質、例えば、抗体、リガンド等によって、確認することができる。例えば、各々に特定的な抗体として以下のものが知られている。 The sequences and structures of the proteins and genes of CD14, CD206, and CXCR4 are known, and each is co-positive, that is, CD14 (+), CD206 (+), and CXCR4 (+) can be examined by a known method. .. The presence of the proteins of CD14, CD206, CXCR4, without limitation, can be confirmed, for example, by a substance that specifically binds to these proteins, for example, an antibody, a ligand, or the like. For example, the following are known as specific antibodies for each.
ＣＤ１４特異的な抗体： ＡＰＣ－Ｃｙ７－抗ヒトＣＤ１４抗体（ＢｉｏＬｅｇｅｎｄ社製）
ＣＤ２０６特異的な抗体：ＰＥ－ｃｙ７－抗ヒトＣＤ２０６抗体（ＢｉｏＬｅｇｅｎｄ社製）
ＣＸＣＲ４特異的な抗体：ＡＰＣ－抗ヒトＣＸＣＲ４抗体（ＢｉｏＬｅｇｅｎｄ社製）
これらの抗体を用いて例えば、蛍光抗体法等によりこれらのタンパク質の存否を確認し、例えば、フローサイトメトリー、マイクロビーズ標識による磁気回収等によりＣＤ１４（＋）、ＣＤ２０６（＋）、ＣＸＣＲ４（＋）の細胞をソートすることができる。
あるいは、ポリメラーゼ連鎖反応（ＰＣＲ）、ＲＮＡシーケンス（ＲＮＡ－Ｓｅｑ） 等の方法を用いて、ＣＤ１４、ＣＤ２０６、ＣＸＣＲ４の核酸の存否を確認してもよい。
CD14-specific antibody: APC-Cy7-anti-human CD14 antibody (manufactured by BioLegend)
CD206-specific antibody: PE-cy7-anti-human CD206 antibody (manufactured by BioLegend)
CXCR4-specific antibody: APC-anti-human CXCR4 antibody (manufactured by BioLegend)
Using these antibodies, for example, the presence or absence of these proteins is confirmed by a fluorescent antibody method or the like, and for example, CD14 (+), CD206 (+), CXCR4 (+) are confirmed by flow cytometry, magnetic recovery by microbead labeling, or the like. Cells can be sorted.
Alternatively, the presence or absence of the nucleic acids of CD14, CD206, and CXCR4 may be confirmed by using a method such as polymerase chain reaction (PCR) or RNA sequencing (RNA-Seq).
２．細胞群
本発明は、一態様において、細胞群に関する。
2. 2. Cell Group The present invention relates to a cell group in one embodiment.
上記細胞群は、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞を含む。一態様において（即ち、非限定的に）上記細胞群は、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞を４０％以上、５０％以上、６０％以上、７０％以上、８０％以上、９０％以上、９５％以上、９８％以上含む。 The cell group includes cells that are CD14 (+) CD206 (+) CXCR4 (+). In one embodiment (ie, but not limited to), the cell population comprises 40% or more, 50% or more, 60% or more, 70% or more, 80% of cells that are CD14 (+) CD206 (+) CXCR4 (+). Includes 90% or more, 95% or more, and 98% or more.
細胞群中に含まれるＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞の割合は、公知の方法によって調べることができる。例えば、フローサイトメトリーにより細胞群がＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞を４０％以上含む、と判断できる。 The proportion of cells that are CD14 (+) CD206 (+) CXCR4 (+) contained in the cell group can be examined by a known method. For example, it can be determined by flow cytometry that the cell group contains 40% or more of cells having CD14 (+) CD206 (+) CXCR4 (+).
一態様において（即ち、非限定的に）ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（－）の細胞は４０％以下、３０％以下、２０％以下、１０％以下、５％以下である。ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（－）の細胞の割合は、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞の割合に関して上述したのと同様の方法によって行うことができる。 In one embodiment (ie, but not limited to), the cells of CD14 (+) CD206 (+) CXCR4 (−) are 40% or less, 30% or less, 20% or less, 10% or less, 5% or less. The proportion of cells of CD14 (+) CD206 (+) CXCR4 (−) can be determined by the same method as described above with respect to the proportion of cells of CD14 (+) CD206 (+) CXCR4 (+).
３．細胞又は細胞群の取得方法
上記ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞を含む細胞群の取得方法は特に限定されない。
3. 3. Method for Obtaining Cell or Cell Group The method for obtaining a cell group including the above-mentioned CD14 (+) CD206 (+) CXCR4 (+) cell and CD14 (+) CD206 (+) CXCR4 (+) cell is not particularly limited. ..
一態様において、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞を含む細胞群は、骨髄、臍帯血又は末梢血由来の単核球を培養することによって得られる。上記細胞群の取得のために用いられる「単核球」とは、末梢血、骨髄又は臍帯血等に含まれる円形核を持つ細胞の総称で、リンパ球、単球、マクロファージ、血管内皮前駆細胞、造血幹細胞等が含まれる。例えば、動物から骨髄、臍帯血又は末梢血を採取し、それを例えば単核球分離用採血管を用いることにより、又は密度勾配遠心法に付して該分画を抽出することにより単核球が得られる。密度勾配遠心法としては、単核球分画が形成されれば特に限定されない。例えば、Ｈｉｓｔｏｐａｑｕｅ－１０７７（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）を用いてもよい。 In one embodiment, the cell population comprising cells CD14 (+) CD206 (+) CXCR4 (+), CD14 (+) CD206 (+) CXCR4 (+) is derived from bone marrow, cord blood or peripheral blood. Obtained by culturing mononuclear cells. "Monocyte" used for acquisition of the above cell group is a general term for cells having a round nucleus contained in peripheral blood, bone marrow, cord blood, etc., and is a lymphocyte, monocyte, macrophage, vascular endothelial progenitor cell. , Hematopoietic stem cells, etc. are included. For example, mononuclear cells by collecting bone marrow, umbilical cord blood or peripheral blood from an animal and extracting the fraction by, for example, using a blood collection tube for mononuclear cell separation or subject to density gradient centrifugation. Is obtained. The density gradient centrifugation method is not particularly limited as long as a mononuclear ball fraction is formed. For example, Histopaque-1077 (Sigma-Aldrich) may be used.
骨髄、臍帯血又は末梢血が由来する動物種は、特に限定されない。動物種には、虚血性疾患、炎症性疾患、又は難治性創傷等の疾患に対する細胞移植療法が適用されるヒトを含む哺乳動物一般が含まれる。臨床応用という目的に鑑みれば、好ましくはヒトである。 The animal species from which bone marrow, cord blood or peripheral blood is derived is not particularly limited. Animal species include mammals in general, including humans, to whom cell transplantation therapy is applied for diseases such as ischemic disease, inflammatory disease, or refractory wound. In view of the purpose of clinical application, it is preferably human.
一態様において、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞は、（ｉ）骨髄、臍帯血又は末梢血由来の単核球を、幹細胞因子、インタ一ロイキン６、ＦＭＳ様チロシンキナーゼ３リガンド、トロンボポエチン及び血管内皮細胞増殖因子の５種類の因子もしくはこの５種類の因子から選択される３因子又は４因子、並びに、血清を含む培地もしくは血清を含まない培地中で培養し、そして（ｉｉ）ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）選択的に回収することにより得られる。 In one embodiment, the cells CD14 (+) CD206 (+) CXCR4 (+) are (i) mononuclear cells from bone marrow, cord blood or peripheral blood, stem cell factor, interleukin 6, FMS-like tyrosine kinase. Cultivated in 3 ligands, 5 factors of thrombopoietin and vascular endothelial growth factor or 3 or 4 factors selected from these 5 factors, and serum-containing or serum-free medium, and ( ii) CD14 (+) CD206 (+) CXCR4 (+) Obtained by selective recovery.
また、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞を含む細胞群は、骨髄、臍帯血又は末梢血由来の単核球を、幹細胞因子、インタ一ロイキン６、ＦＭＳ様チロシンキナーゼ３リガンド、トロンボポエチン及び血管内皮細胞増殖因子の５種類の因子もしくはこの５種類の因子から選択される３因子又は４因子、並びに、血清を含む培地もしくは血清を含まない培地中で培養することで得られる。 In addition, the cell group containing cells of CD14 (+) CD206 (+) CXCR4 (+) can be mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood, stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 Obtained by culturing in 5 factors of ligand, thrombopoietin and vascular endothelial growth factor, or 3 or 4 factors selected from these 5 factors, and in a medium containing or not containing serum. ..
幹細胞因子（ＳＣＦ）は、２４８個のアミノ酸からなる分子量約３０，０００の糖タンパク質である。選択的スプライシングにより可溶型と膜結合型が存在するが、上記細胞群の取得のためにＳＣＦは単核球の培養に有用である限りいずれのタイプのＳＣＦでもよい。好ましくは可溶型である。ＳＣＦの由来等は特に限定されない。非限定的に、安定した供給が見込まれる組換え体が好ましく、特に好ましくはヒト組換え体である。商業的に入手可能なものが知られている。培養培地中のＳＣＦの濃度は、用いるＳＣＦの種類によっても異なり、単核球の培養に有用である限り特に限定されない。ヒト組換えＳＣＦの場合であれば、非限定的に、例えば１０～１０００ｎｇ／ｍＬ、好ましくは５０～５００ｎｇ／ｍＬ、より好ましくは約１００ｎｇ／ｍＬである。 Stem cell factor (SCF) is a glycoprotein consisting of 248 amino acids and having a molecular weight of about 30,000. There are soluble and membrane-bound types by alternative splicing, but the SCF may be any type of SCF as long as it is useful for culturing mononuclear cells for the acquisition of the above cell population. It is preferably a soluble type. The origin of SCF is not particularly limited. A recombinant that is expected to have a stable supply is preferable without limitation, and a human recombinant is particularly preferable. Commercially available ones are known. The concentration of SCF in the culture medium also depends on the type of SCF used and is not particularly limited as long as it is useful for culturing mononuclear cells. In the case of human recombinant SCF, it is not limited, for example, 10 to 1000 ng / mL, preferably 50 to 500 ng / mL, and more preferably about 100 ng / mL.
インターロイキン６（ＩＬ－６）は、Ｂ細胞の抗体産生細胞への最終分化を誘導する因子として単離された分子量２１万の糖タンパク質である。ＩＬ－６は、一般に、免疫応答、造血系や神経系細胞の増殖分化、急性期反応等に関与することが知られている。上記細胞群の取得のために用いるＩＬ－６は、特に限定されず、適宜選択される。ヒトの単核球の培養に用いる場合には、ヒトＩＬ－６が好ましく、安定した供給が見込まれる組換え体が特に好ましい。商業的に入手可能なものが知られている。培養培地中のＩＬ－６の濃度は、用いるＩＬ－６の種類によっても異なり、単核球の培養に有用である限り特に限定されない。ヒト組換えＩＬ－６の場合であれば、非限定的に、例えば１～５００ｎｇ／ｍＬ、好ましくは５～１００ｎｇ／ｍＬ、より好ましくは約２０ｎｇ／ｍＬである。 Interleukin 6 (IL-6) is a glycoprotein having a molecular weight of 210,000 isolated as a factor that induces the final differentiation of B cells into antibody-producing cells. IL-6 is generally known to be involved in immune response, proliferation and differentiation of hematopoietic and nervous system cells, acute phase reaction and the like. The IL-6 used for acquiring the cell group is not particularly limited and is appropriately selected. When used for culturing human mononuclear cells, human IL-6 is preferable, and a recombinant that is expected to have a stable supply is particularly preferable. Commercially available ones are known. The concentration of IL-6 in the culture medium also varies depending on the type of IL-6 used, and is not particularly limited as long as it is useful for culturing mononuclear cells. In the case of human recombinant IL-6, it is not limited, for example, 1 to 500 ng / mL, preferably 5 to 100 ng / mL, and more preferably about 20 ng / mL.
ＦＭＳ様チロシンキナーゼ３リガンド（ＦＬ）は、初期造血制御において重要な役目を担う受容体型チロシンキナーゼのリガンドとして知られている。いくつかの選択的スプライシングによる産物が知られているが、造血系幹細胞の増殖を刺激するという報告がある。上記細胞群の取得のために用いるＦＬは、単核球の培養に有用である限り、いずれのタイプのＦＬであってもよい。商業的に入手可能なものが知られている。培養培地中のＦＬの濃度は、用いるＦＬの種類によっても異なり、単核球の培養に有用である限り特に限定されない。ヒト組換えＦｌｔ－３リガンドの場合であれば、非限定的に、例えば１０～１０００ｎｇ／ｍＬ、好ましくは５０～５００ｎｇ／ｍＬ、より好ましくは約１００ｎｇ／ｍＬである。 The FMS-like tyrosine kinase 3 ligand (FL) is known as a ligand for receptor tyrosine kinase that plays an important role in the regulation of early hematopoiesis. Although some alternative splicing products are known, they have been reported to stimulate the proliferation of hematopoietic stem cells. The FL used for acquiring the cell group may be any type of FL as long as it is useful for culturing mononuclear cells. Commercially available ones are known. The concentration of FL in the culture medium also varies depending on the type of FL used, and is not particularly limited as long as it is useful for culturing mononuclear cells. In the case of human recombinant Flt-3 ligand, it is not limited, for example, 10 to 1000 ng / mL, preferably 50 to 500 ng / mL, and more preferably about 100 ng / mL.
トロンボポエチン（ＴＰＯ）は、造血系サイトカインの一種であり、造血幹細胞から巨核球が作られる過程に特異的に作用し、巨核球の産生を促進することが知られている。上記細胞群の取得のために用いるＴＰＯの由来等は特に限定されない。安定した供給が見込まれる組換え体が好ましく、特に好ましくはヒト組換え体である。商業的に入手可能なものが知られている。培養培地中のＴＰＯの濃度は、用いるＴＰＯの種類によっても異なり、単核球の培養に有用である限り特に限定されないが、ヒト組換えＴＰＯの場合であれば、非限定的に、例えば１～５００ｎｇ／ｍＬ、好ましくは５～１００ｎｇ／ｍＬ、より好ましくは約２０ｎｇ／ｍＬである。 Thrombopoietin (TPO) is a type of hematopoietic cytokine and is known to act specifically on the process of making megakaryocytes from hematopoietic stem cells and promote the production of megakaryocytes. The origin of TPO used for acquiring the above cell group is not particularly limited. A recombinant that is expected to have a stable supply is preferable, and a human recombinant is particularly preferable. Commercially available ones are known. The concentration of TPO in the culture medium varies depending on the type of TPO used and is not particularly limited as long as it is useful for culturing mononuclear cells, but in the case of human recombinant TPO, it is not limited, for example, 1 to 1. It is 500 ng / mL, preferably 5 to 100 ng / mL, more preferably about 20 ng / mL.
血管内皮細胞増殖因子（ＶＥＧＦ）は、血管内皮前駆細胞（ＥＰＣ）に特異的に作用する増殖因子であり、主に血管周囲の細胞で産生されることが知られている。選択的スプライシングによってサイズの異なる数種のＶＥＧＦタンパク質が産生されるが、上記細胞群の取得のために用いるＶＥＧＦはＥＰＣのコロニー形成を可能にする限りいずれのタイプのＶＥＧＦでもよい。好ましくはＶＥＧＦ１６５である。ＶＥＧＦの由来等は特に限定されない。安定した供給が見込まれる組換え体が好ましく、特に好ましくはヒト組換え体である。商業的に入手可能なものが知られている。培養培地中のＶＥＧＦの濃度は、用いるＶＥＧＦの種類によっても異なり、単核球の培養に有用である限り特に限定されな。ヒト組換えＶＥＧＦ１６５の場合であれば、非限定的に、例えば約５～５００ｎｇ／ｍＬ、好ましくは約２０～１００ｎｇ／ｍＬ、より好ましくは約５０ｎｇ／ｍＬである。 Vascular Endothelial Growth Factor (VEGF) is a growth factor that specifically acts on vascular endothelial progenitor cells (EPCs) and is known to be produced mainly by cells around blood vessels. Although alternative splicing produces several VEGF proteins of different sizes, the VEGF used to obtain the cell population may be any type of VEGF as long as it allows EPC colonization. VEGF165 is preferred. The origin of VEGF is not particularly limited. A recombinant that is expected to have a stable supply is preferable, and a human recombinant is particularly preferable. Commercially available ones are known. The concentration of VEGF in the culture medium also depends on the type of VEGF used and is not particularly limited as long as it is useful for culturing mononuclear cells. In the case of human recombinant VEGF165, it is not limited, for example, about 5 to 500 ng / mL, preferably about 20 to 100 ng / mL, and more preferably about 50 ng / mL.
単核球の培養に用いる培養培地に添加される各種因子はまた、非限定的に、単核球が由来する動物と同種の動物に由来する因子で統一してもよい。このように単核球及び各種因子の由来を統一することで、同種異系移植等の同種移植に好適な細胞培養物が得られる。また、細胞移植が意図される個体由来の単核球を用いることで、同種同系移植に好適な細胞培養物を得ることも可能である。 The various factors added to the culture medium used for culturing mononuclear cells may also be unified with factors derived from animals of the same species as the animals from which the mononuclear cells are derived, without limitation. By unifying the origins of mononuclear cells and various factors in this way, cell cultures suitable for allogeneic transplantation such as allogeneic transplantation can be obtained. It is also possible to obtain a cell culture suitable for allogeneic transplantation by using mononuclear cells derived from an individual intended for cell transplantation.
上記した各成分は培養培地で所定の濃度に溶解するか、あるいはあらかじめ各成分の濃縮液（ストック溶液）を調製し、培養培地で所定の濃度に希釈することによって、単核球を培養するための培養培地を調製することができる。例えば市販の培養培地に必要な成分を所定の濃度となるよう溶解した後、濾過滅菌等により滅菌するか、あるいは濾過滅菌等により滅菌したストック溶液を無菌的に市販の培養培地に添加、希釈することによって培養培地を調製することができる。濾過滅菌は当分野で通常実施されている方法に準じて行うことができ、例えば０．２２μｍや０．４５μｍのミリポアフィルター等を用いて行う。 To cultivate mononuclear cells by dissolving each of the above components in a culture medium to a predetermined concentration, or by preparing a concentrated solution (stock solution) of each component in advance and diluting it to a predetermined concentration in the culture medium. Culture medium can be prepared. For example, after dissolving the necessary components in a commercially available culture medium to a predetermined concentration, sterilize by filtration sterilization or the like, or aseptically add and dilute the stock solution sterilized by filtration sterilization or the like to the commercially available culture medium. This makes it possible to prepare a culture medium. Filtration sterilization can be performed according to a method usually practiced in the art, for example, using a 0.22 μm or 0.45 μm millipore filter or the like.
本発明で用いられる「培養培地」は、当分野で通常用いられている培地を利用することができ、例えば造血幹細胞の増殖用培地として知られている培養地を用いることができる。培養培地として用いられる基礎培地としては、例えば、Ｓｔｅｍｌｉｎｅ ＩＩ、ＤＭＥＭ、ＭＥＭ、ＩＭＤＭ、ＲＰＭＩ、ＳＣＧＭ、ＥＢＭ等が挙げられる。 As the "culture medium" used in the present invention, a medium usually used in the art can be used, and for example, a culture medium known as a growth medium for hematopoietic stem cells can be used. Examples of the basal medium used as the culture medium include Stemline II, DMEM, MEM, IMDM, RPMI, SCGM, EBM and the like.
上記細胞、細胞群を取得するために、単核球を培養するための培地は、幹細胞因子、インタ一ロイキン６、ＦＭＳ様チロシンキナーゼ３リガンド、トロンボポエチン及び血管内皮細胞増殖因子の５種類の因子、もしくはこの５種類の因子から選択される３因子又は４因子の他に、血清を含む培地もしくは含まない培地、を含む。 The medium for culturing mononuclear cells to obtain the above cells and cell groups is a stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor. Alternatively, in addition to the 3 or 4 factors selected from these 5 types of factors, a medium containing or not containing serum is included.
血清の種類は特に限定されない。非限定的に、一態様において、血清は、ウシ又はヒトの血清である。一態様において、血清は、ウシとヒトの血清両方を含んでもよい。一態様において、血清は、ウシ胎児血清である。培地中における血清の濃度も特に限定されない。一態様において、血清は培地中に、０．１容量％以上、０．３容量％以上、０．５容量％以上含まれる。培地中の血清の濃度の上限は特にない。一態様において、３０容量％以内、２０容量％以内、１０容量％以内、５容量％以内である。非限定的に、血清は、培地中に０．１容量％以上２０容量％の濃度、培地中に０．５容量％以上１０容量％の濃度で含まれる。 The type of serum is not particularly limited. In one embodiment, without limitation, the serum is bovine or human serum. In one embodiment, the serum may contain both bovine and human serum. In one embodiment, the serum is fetal bovine serum. The concentration of serum in the medium is also not particularly limited. In one embodiment, the serum is contained in the medium in an amount of 0.1% by volume or more, 0.3% by volume or more, and 0.5% by volume or more. There is no particular upper limit to the concentration of serum in the medium. In one embodiment, it is within 30% by volume, within 20% by volume, within 10% by volume, and within 5% by volume. Non-limitingly, serum is contained in the medium at a concentration of 0.1% by volume or more and 20% by volume, and in the medium at a concentration of 0.5% by volume or more and 10% by volume.
単核球培養は、単核球を含有する細胞懸濁液を、上述した因子及び血清を含む培地もしくは血清を含まない培地に添加することにより行われる。細胞懸濁液としてはまた、単核球を含有する体液自体（例えば、骨髄液、臍帯血、末梢血）を用いることもできる。単核球の培養条件は特に限定されず、通常当分野で実施される条件で実施することができる。非限定的に、例えば、５％ＣＯ２雰囲気下、約３７℃で培養される。培養期間は、非限定的に例えば、３日間以上であり、５日間以内、６日間以内、７日間以内、１０日間以内である。例えば、３日間－１０日間、３日間－６日間である。単核球の培地中の濃度は、単核球の培養を可能とする限り特に限定されないが、例えば約０．１～１０×１０６細胞／ｍｌ、より好ましくは約０．５～５×１０６細胞／ｍｌである。 Mononuclear cell culture is performed by adding a cell suspension containing mononuclear cells to a medium containing the above-mentioned factors and serum or a medium containing no serum. As the cell suspension, the body fluid itself containing mononuclear cells (for example, bone marrow fluid, umbilical cord blood, peripheral blood) can also be used. The culture conditions for mononuclear cells are not particularly limited, and can be carried out under the conditions normally carried out in the art. Non-limitingly, for example, it is cultured at about 37 ° C. in a 5% CO 2 atmosphere. The culture period is not limited, for example, 3 days or more, 5 days or less, 6 days or less, 7 days or less, and 10 days or less. For example, 3 days-10 days, 3 days-6 days. The concentration of the mononuclear cells in the medium is not particularly limited as long as the mononuclear cells can be cultured, but for example, about 0.1 to 10 × 106 cells / ml, more preferably about 0.5 to 5 × 10. 6 cells / ml.
「ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）選択的に回収する」ことは、例えば「１．細胞」で説明したＣＤ１４、ＣＤ２０６、ＣＸＣＲ４の存在確認するための方法を適宜利用することにより、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）細胞、即ち、ＣＤ１４、ＣＤ２０６、ＣＸＣＲ４が存在する細胞を選択的に回収（ソート）することにより行うことができる。例えば、これらのタンパク質に特異的に結合する物質、例えば、抗体、リガンド等を利用したフローサイトメトリー、マイクロビーズ標識による磁気回収等が利用できる。
上述した方法は、上記細胞、細胞群を得るための一例である。
"Selective recovery of CD14 (+) CD206 (+) CXCR4 (+)" means, for example, by appropriately using the method for confirming the presence of CD14, CD206, CXCR4 described in "1. Cell". This can be done by selectively collecting (sorting) CD14 (+) CD206 (+) CXCR4 (+) cells, that is, cells in which CD14, CD206, and CXCR4 are present. For example, flow cytometry using a substance that specifically binds to these proteins, for example, an antibody, a ligand, or the like, magnetic recovery by microbead labeling, or the like can be used.
The above-mentioned method is an example for obtaining the above-mentioned cells and cell groups.
４．治療用組成物
本発明は、一態様において、虚血性疾患、炎症性疾患又は難治療性創傷の治療用組成物に関する。本発明の治療用組成物は、「１．細胞」「２．細胞群」で説明した細胞、細胞群を含む。細胞、細胞群は、治療用組成物を使用する対象に由来しても（自家）、治療用組成物を使用する対象以外に由来しても（他家）よい。
4. Therapeutic Composition The present invention relates, in one aspect, to a therapeutic composition for an ischemic disease, an inflammatory disease or a refractory wound. The therapeutic composition of the present invention includes the cells and cell groups described in "1. Cells" and "2. Cell Groups". The cells and cell groups may be derived from a subject for which the therapeutic composition is used (autologous) or may be derived from a subject other than the subject for which the therapeutic composition is used (allogeneic).
「虚血性疾患」は、血量の減少によって組織内の血流が下がり、細胞の変性、萎縮、線維化などの組織障害が生じることによって起こる疾患である。虚血はその原因により、閉塞性虚血、圧迫性虚血、痙攣性虚血、代償性虚血に大別される。虚血が持続すると細胞の変性、萎縮、線維化が生じる。「虚血性疾患」には、四肢虚血、虚血性潰瘍、虚血性心疾患、脳梗塞、虚血性腸炎、虚血性肝炎、虚血性腎障害等が含まれる。「脳梗塞」（又は脳軟化症）は、脳を栄養する動脈の閉塞、又は狭窄のため、脳虚血を来たし、脳組織が酸素、又は栄養の不足のため壊死、又は壊死に近い状態になる事をいう、脳血栓及び脳塞栓に分類される。「四肢虚血」は、手足に血液を供給する動脈が狭窄又は閉塞する疾患であり、この閉塞性動脈硬化症が重症化したり、膠原病により、重症の四肢虚血となり、疼痛などの症状及び難治性の潰瘍が生じ、最悪の場合、切断が必要になる場合もある。非限定的に、四肢は一態様において下肢である。 "Ischemic disease" is a disease caused by a decrease in blood flow, which reduces blood flow in a tissue and causes tissue damage such as cell degeneration, atrophy, and fibrosis. Ischemia is roughly classified into obstructive ischemia, compressive ischemia, spasmodic ischemia, and compensatory ischemia depending on the cause. Persistent ischemia results in cell degeneration, atrophy, and fibrosis. The "ischemic disease" includes limb ischemia, ischemic ulcer, ischemic heart disease, cerebral infarction, ischemic enteritis, ischemic hepatitis, ischemic nephropathy and the like. "Cerebral infarction" (or cerebral infarction) causes cerebral ischemia due to obstruction or stenosis of the arteries that feed the brain, and the brain tissue becomes necrotic or near necrosis due to lack of oxygen or nutrients. It is classified into cerebral thrombosis and cerebral infarction. "Ischemia of the limbs" is a disease in which the arteries that supply blood to the limbs are narrowed or occluded. Intractable ulcers develop and, in the worst case, may require amputation. Not limited, the limbs are, in one aspect, the lower limbs.
非限定的に、一態様において、虚血性疾患は、四肢虚血である。虚血性疾患は、潰瘍を伴う四肢虚血である。
「炎症性疾患」は、なんらかの原因により組織損傷などの異常が生じることで症状を起こす疾患の総称である。炎症性疾患には、クローン病、肝硬変、肝炎、潰瘍性大腸炎、炎症性腸疾患等が含まれる。
In one embodiment, but not limited, the ischemic disease is limb ischemia. The ischemic disease is limb ischemia with ulcers.
"Inflammatory disease" is a general term for diseases that cause symptoms due to abnormalities such as tissue damage caused by some cause. Inflammatory diseases include Crohn's disease, cirrhosis, hepatitis, ulcerative colitis, inflammatory bowel disease and the like.
上記治療用組成物は、血管新生及び／又は創傷治癒を促進する。よって、血管新生及び／又は創傷治癒により治療できる疾患に適用することができる。「血管新生及び／又は創傷治癒により治療できる疾患」としては、例えば、虚血性疾患（例えば、心筋梗塞、狭心症等の虚血性心疾患、下肢虚血性動脈硬化症等の下肢虚血、バージャー（Ｂｕｒｇｅｒ）病）、血管損傷が挙げられる。また、皮膚潰瘍等の創傷を治癒するため、あるいは人工血管の作製に使用できる。治療用組成物の適用の効果は、自体公知の方法により確認できる。例えば血管新生により治療できる疾患が下肢虚血性疾患である場合には、移植後の治療効果を例えば下肢血流量及び壊死改善率を調べることによって評価することができる。血流量の増加の測定はレーザドップラーイメージング解析の値を測定することによって行うことができる。また壊死改善率はリムサルベージスコアとして目視により測定することができる。 The therapeutic composition promotes angiogenesis and / or wound healing. Therefore, it can be applied to diseases that can be treated by angiogenesis and / or wound healing. "Disease that can be treated by angiogenesis and / or wound healing" includes, for example, ischemic disease (for example, ischemic heart disease such as myocardial infarction and angina, lower limb ischemia such as lower limb ischemic arteriosclerosis, and Burger. (Burger) disease), vascular injury. It can also be used to heal wounds such as skin ulcers or to create artificial blood vessels. The effect of applying the therapeutic composition can be confirmed by a method known per se. For example, when the disease that can be treated by angiogenesis is a lower limb ischemic disease, the therapeutic effect after transplantation can be evaluated, for example, by examining the lower limb blood flow and the necrosis improvement rate. The increase in blood flow can be measured by measuring the values of the laser Doppler imaging analysis. The necrosis improvement rate can be visually measured as a limb salvage score.
上記治療用組成物の使用態様は特に限定されない。有効成分たる細胞群又は細胞群そのものであってもよいし、細胞又は細胞群を液状媒体に懸濁したものであってもよい。液体媒体はヒトに注入可能な液体であればいずれであってもよく、例えば、等張電解質輸液やリン酸緩衝液や生理食塩水あるいは無血清培地であるＤＭＥＭなども利用可能である。また液体媒体にはアルブミンなど細胞の生存に好ましい化合物が含まれてもよい。また凍結保存する場合には凍結細胞保存液などで懸濁したものであってもよい。 The mode of use of the therapeutic composition is not particularly limited. It may be a cell group or a cell group itself as an active ingredient, or a cell or a cell group suspended in a liquid medium. The liquid medium may be any liquid that can be injected into humans, and for example, isotonic electrolyte infusion, phosphate buffer, physiological saline, or DMEM, which is a serum-free medium, can be used. The liquid medium may also contain a compound such as albumin that is preferable for cell survival. Further, in the case of cryopreservation, it may be suspended in a frozen cell preservation solution or the like.
上記治療用組成物の適用対象は、虚血性疾患、炎症性疾患又は難治療性創傷の治療が必要である対象であれば、特に限定されない。非限定的に、ヒト、サル、チンパンジー、イヌ、ネコ、ウシ、ウマ、ネズミ、モルモット等が含まれる。 The target of application of the therapeutic composition is not particularly limited as long as it is a subject requiring treatment of ischemic disease, inflammatory disease or intractable wound. Non-limitingly included, humans, monkeys, chimpanzees, dogs, cats, cows, horses, mice, guinea pigs and the like.
４．治療方法等
本発明は、一態様において、「１．細胞」「２．細胞群」で説明した細胞、細胞群を、必要のある対象に適用することを含む、虚血性疾患、炎症性疾患又は難治療性創傷の治療方法に関する。
4. Therapeutic method, etc. In one embodiment, the present invention comprises applying the cells, cell groups described in "1. Cell" and "2. Cell group" to a subject in need, such as ischemic disease, inflammatory disease, or. Regarding the treatment method of intractable wounds.
本発明は、一態様において、「１．細胞」「２．細胞群」で説明した細胞、細胞群の、必要のある対象に適用することを含む、虚血性疾患、炎症性疾患又は難治療性創傷の治療方法への使用、あるいは、虚血性疾患、炎症性疾患又は難治療性創傷の治療用組成物への使用、に関する。 The present invention, in one embodiment, comprises the application of the cells, cell groups described in "1. Cell" and "2. Cell Group" to a subject in need, such as ischemic disease, inflammatory disease or refractory. It relates to a method of treating a wound, or a composition for treating an ischemic disease, an inflammatory disease or a refractory wound.
本発明は、一態様において、虚血性疾患、炎症性疾患又は難治療性創傷の治療方法へ使用するための、「１．細胞」「２．細胞群」で説明した細胞、細胞群に関する。
「虚血性疾患」、「炎症性疾患」又は「難治療性創傷」、「細胞群の適用態様」、「適用対象」等については、「３．治療用組成物」に記載した通りである。
The present invention relates to, in one aspect, the cells and cell groups described in "1. Cells" and "2. Cell Groups" for use in the treatment of ischemic diseases, inflammatory diseases or intractable wounds.
“Ischemic disease”, “inflammatory disease” or “intractable wound”, “application mode of cell group”, “application target” and the like are as described in “3. Therapeutic composition”.
以下、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。当業者は本明細書の記載に基づいて容易に本発明に修飾・変更を加えることができ、それらは本発明の技術的範囲に含まれる。 Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify or modify the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.
実施例１ Ｒｅｍａ細胞の単離
（１）単核球の単離
健常人のボランティアから、ＢＤ バキュテイナ（登録商標）ＣＰＴ（商標）ＢＤ社製）単核球分離用採血管を用いて１００ｍＬの末梢血を採取した。採血後、そのまま採血管を遠心した後、輸送、到着後培養開始までは冷蔵で保管した。単核球及び血漿を含むＣＰＴ（商標）採血管内のゲルバリアより上を遠心管へ回収し、少量のＥＤＴＡ－ＰＢＳでゲルバリア上など採血管内を洗いこみ、同じ遠心管へ回収した。細胞を回収した遠心管をＥＤＴＡ－ＰＢＳでメスアップしたのち、遠心分離（３００ｘｇ、室温、１５分間）し、沈査として細胞を回収した。回収された細胞は、混入する赤血球を除くためＡＣＫ溶血緩衝液（１５ｍＬ／管）（Ｇｉｂｃｏ、Ｔｈｅｒｍｏ－Ｆｉｓｈｅｒ社製）中、室温で５分間インキュベートした。ＡＣＫ溶血緩衝液の組成は、ＮＨ４Ｃｌ ８，２９０ｍｇ／ｌ；ＫＨＣＯ３ １，０００ｍｇ／ｌ；ＥＤＴＡ．Ｎａ２・２Ｈ２Ｏ ３７ｍｇ／ｌである。その後、ＥＤＴＡ－ＰＢＳでメスアップし、遠心分離（２００ｘｇ、室温、１０分間）又は（１００ｘｇ、室温、１５分間）で細胞を回収する操作を２回繰り返した。
Example 1 Isolation of Rema cells (1) Isolation of mononuclear cells From healthy volunteers, BD Vacutainer (registered trademark) CPT (trademark) manufactured by BD, Inc., 100 mL peripheral using a mononuclear cell separation blood collection tube Blood was collected. After blood collection, the blood collection tube was centrifuged as it was, and then transported and stored in a refrigerator until the start of culture after arrival. The area above the gel barrier in the CPT ™ blood collection tube containing mononuclear cells and plasma was collected in a centrifuge tube, and the inside of the blood collection tube such as on the gel barrier was washed with a small amount of EDTA-PBS and collected in the same centrifuge tube. The centrifuge tube from which the cells were collected was measured up with EDTA-PBS, and then centrifuged (300 xg, room temperature, 15 minutes), and the cells were collected as a sediment. Collected cells were incubated in ACK hemolytic buffer (15 mL / tube) (Gibco, Thermo-Fisher) for 5 minutes at room temperature to remove contaminating erythrocytes. The composition of the ACK hemolytic buffer is NH 4 Cl 8,290 mg / l; KHCO 3 1,000 mg / l; EDTA. Na 2.2H 2 O is 37 mg / l. Then, the operation of measuring up with EDTA-PBS and collecting cells by centrifugation (200 xg, room temperature, 10 minutes) or (100 xg, room temperature, 15 minutes) was repeated twice.
（２）無血清培地での培養
回収した単核球細胞を、１ｍＬの増殖培地に懸濁し、その一部を使用してトリパンブルーを用いた細胞数計数を行った。下記に示すいくつかの増殖培地で培養した。培地１は、５０ｎｇ／ｍｌ ＶＥＧＦ、１００ｎｇ／ｍｌ ＳＣＦ、１００ｎｇ／ｍｌ Ｆｌｔ－３リガンド、２０ｎｇ／ｍｌ ＴＰＯ、２０ｎｇ／ｍｌ及びＩＬ－６、１００単位／ｍＬの５種類の因子、並びに、ペニシリン、１００μｇ／ｍＬ ストレプトマイシンを添加した ｓｔｅｍｌｉｎｅ（登録商標） ＩＩ Ｈｅｍａｔｏｐｏｉｅｔｉｃ Ｓｔｅｍ Ｃｅｌｌ Ｅｘｐａｎｓｉｏｎ Ｍｅｄｉｕｍ（シグマ－アルドリッチ社製、Ｃａｔ Ｎｏ．Ｓ０１９２を用いた。
(2) Culture in serum-free medium The recovered mononuclear cells were suspended in 1 mL of growth medium, and a part of the cells was used to count the number of cells using trypan blue. The cells were cultured in several growth media shown below. Medium 1 contains 5 factors: 50 ng / ml VEGF, 100 ng / ml SCF, 100 ng / ml Flt-3 ligand, 20 ng / ml TPO, 20 ng / ml and IL-6, 100 units / mL, and penicillin, 100 μg. / ML Stemline® II Hematopoietic Stem Cell Expansion Medium (manufactured by Sigma-Aldrich, Cat No. S0192) to which streptomycin was added was used.
得られた細胞増殖培地中で、細胞濃度を１ｘ１０６／ｍＬに調製し、６穴培養プレートの各ウェルに２ｍＬずつ播種した。通常の培養条件（３７℃、５％ＣＯ２）で５日間培養した。５日間の培養終了後、遠心管に回収し、２５０ｘｇ（～１０００ｒｐｍ）で７－１０分間、遠心洗浄を３回実施した。遠心後、細胞数計測、生存率測定、ＱＣ（確認）試験用のサンプルを除き、残りの細胞はＰｌａｓｍａＬｙｔｅ Ａ、２．５％ ヒトアルブミン血清に懸濁し、８ｘ１０５細胞／ｍＬの濃度になるように調整した。 In the obtained cell proliferation medium, the cell concentration was adjusted to 1x10 6 / mL, and 2 mL was seeded in each well of the 6-well culture plate. The cells were cultured under normal culture conditions (37 ° C., 5% CO 2 ) for 5 days. After 5 days of culturing, the cells were collected in a centrifuge tube and centrifuged at 250 xg (~ 1000 rpm) for 7-10 minutes three times. After centrifugation, except for cell count, viability, and QC (confirmation) test samples, the remaining cells were suspended in PlasmaLyte A, 2.5% human albumin serum to a concentration of 8x10 5 cells / mL. Adjusted to.
（３）フローサイトメトリー解析
本実施例では、上記の培養によって得られた細胞群について、その中のＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）細胞およびＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（―）細胞の含有量を明らかにするため、フローサイトメトリー解析を行った。
(3) Flow cytometry analysis In this example, the CD14 (+) CD206 (+) CXCR4 (+) cells and the CD14 (+) CD206 (+) CXCR4 (+) CXCR4 (+) CXCR4 (+) CXCR4 (+) CXCR4 (+) CXCR4 (+) cells in the cell group obtained by the above culture in this example. ―) Flow cytometric analysis was performed to clarify the cell content.
ＦＡＣＳ緩衝液（組成：２％ ＦＢＳを添加した２ｍＭ ＥＤＴＡ－ＰＢＳ）中に懸濁した細胞（１．５×１０６細胞／３００μＬ－ＦＡＣＳ緩衝液）にＦＣブロッキング試薬（ミルテニィ社製）を１０μＬ添加し、４℃で３０分間インキュベートした。インキュベート後の細胞群を、染色反応用チューブに等量ずつ分注した（１００μＬ／チューブ×３チューブ）。各アリコートに各一次抗体を２μＬ添加し、４℃で２０分間培養した。その後１ｍＬのＦＡＣＳ緩衝液で２回洗浄し、染色された細胞をＦＡＣＳ緩衝液中に懸濁した（５×１０５細胞／２００～３００μＬ－ＦＡＣＳ緩衝液）。フローサイトメトリー計測は、ＢＤ ＦＡＣＳＡｒｉａ（商標） ＩＩＩセルソーター（ＢＤ社製）を用いて行った。 Add 10 μL of FC blocking reagent (manufactured by Milteny) to cells (1.5 × 106 cells / 300 μL-FACS buffer) suspended in FACS buffer (composition: 2 mM EDTA-PBS supplemented with 2% FBS). And incubated at 4 ° C. for 30 minutes. The cells after incubation were dispensed into the staining reaction tubes in equal amounts (100 μL / tube × 3 tubes). 2 μL of each primary antibody was added to each aliquot and cultured at 4 ° C. for 20 minutes. Then, the cells were washed twice with 1 mL of FACS buffer, and the stained cells were suspended in FACS buffer (5 × 10 5 cells / 200 to 300 μL-FACS buffer). Flow cytometry measurements were performed using a BD FACSAria ™ III cell sorter (manufactured by BD).
具体的には、得られた細胞群における各細胞表面マーカーに対する抗体を用いて各細胞表面マーカーの発現を調べた。なお、各細胞表面マーカーに対する抗体は、いずれも下記の市販のものを使用した。 Specifically, the expression of each cell surface marker was examined using an antibody against each cell surface marker in the obtained cell group. As the antibody against each cell surface marker, the following commercially available ones were used.
抗ＣＤ２０６抗体：ＰＥ／Ｃｙ７標識抗ヒトＣＤ２０６ （ＭＭＲ）抗体（ＢｉｏＬｅｇｅｎｄ社製）；
抗ＣＸＣＲ４抗体：ＡＰＣ標識抗ヒトＣＤ１８４（ＣＸＣＲ４）抗体（ＢＤ社製）、
抗ＣＤ１４抗体： ＡＰＣ－Ｃｙ７－抗ヒトＣＤ１４抗体（ＢｉｏＬｅｇｅｎｄ社製）。
ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）である細胞を、「Ｒｅｍａ細胞」、ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（－）である細胞を、「Ｎｏｎ－Ｒｅｍａ細胞」、と呼称する場合がある。
本細胞群にはＲｅｍａ細胞が３３．３４％、Ｎｏｎ－Ｒｅｍａ細胞が２８．１７％含まれてた。
Anti-CD206 antibody: PE / Cy7-labeled anti-human CD206 (MMR) antibody (manufactured by BioLegend);
Anti-CXCR4 antibody: APC-labeled anti-human CD184 (CXCR4) antibody (manufactured by BD),
Anti-CD14 antibody: APC-Cy7-anti-human CD14 antibody (manufactured by BioLegend).
When the cell which is CD14 (+) CD206 (+) CXCR4 (+) is referred to as "Rema cell" and the cell which is CD14 (+) CD206 (+) CXCR4 (-) is referred to as "Non-Rema cell". There is.
This cell group contained 33.34% of Rema cells and 28.17% of Non-Rema cells.
（４）Ｒｅｍａ細胞、Ｎｏｎ－Ｒｅｍａ細胞の単離
本実施例において、上記の方法において取得した単核球より培養した培養物よりＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）であるＲｅｍａ細胞とＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（－）であるＮｏｎ－Ｒｅｍａ細胞を分離した。
(4) Isolation of Rema cells and Non-Rema cells In this example, the cultures cultured from mononuclear cells obtained by the above method were used as CD14 (+) CD206 (+) CXCR4 (+) Rema cells. Non-Rema cells, which are CD14 (+) and CD206 (+) CXCR4 (−), were isolated.
細胞数計測後、５０ｍＬ遠心管に１×１０７細胞／３００μＬとなるようＦＡＣＳ緩衝液（組成：２％ ＦＢＳを添加した２ｍＭ ＥＤＴＡ－ＰＢＳ）で懸濁調製した。ついで、１×１０７細胞／２０μＬ ＦｃＲブロッキング試薬（ミルテニィバイオテク社製）を添加し、４℃下で１０分間インキュベートした。 After counting the number of cells, the cells were suspended in a 50 mL centrifuge tube with FACS buffer (composition: 2 mM EDTA-PBS supplemented with 2% FBS) so as to have 1 × 107 cells / 300 μL. Then, 1 × 10 7 cells / 20 μL FcR blocking reagent (manufactured by Milteny Biotech) was added and incubated at 4 ° C. for 10 minutes.
ＰＥｃｙ７－抗ヒトＣＤ２０６抗体（ＢｉｏＬｅｇｅｎｄ社製）とＡＰＣ－抗ヒトＣＸＣＲ４抗体（ＢｉｏＬｅｇｅｎｄ社製）を各々１×１０７細胞／４００μＬ量添加した。ここにおいて、上記２種の抗体による染色前に、Ｓｏｒｔｉｎｇ設定調整用にアイソタイプ対照、ＣＤ２０６単染色、ＣＸＣＲ４単染色分を各々２～５×１０５細胞ずつ１．５ｍＬチューブに取り分け染色した。 PEcy7-anti-human CD206 antibody (manufactured by BioLegend) and APC-anti-human CXCR4 antibody (manufactured by BioLegend) were added in an amount of 1 × 107 cells / 400 μL, respectively. Here, prior to staining with the above two antibodies, isotype control, CD206 single staining, and CXCR4 single staining were separately stained in 1.5 mL tubes with 2 to 5 × 10 5 cells each for adjusting the Sorting setting.
抗体添加後、４℃下で３０分インキュベートした。ＦＡＣＳ緩衝液を足して５０ｍＬとし、遠心した（２５０Ｇ×１０分、４℃）。上清を吸引後、ＦＡＣＳ緩衝液で１×１０７細胞／３ｍＬに懸濁し、セルストレーナー付き５ｍＬチューブ（フローサイトメーター用、Ｃｏｒｎｉｎｇ社製，米国、ＮＹ）へ移し、調整した。フローサイトメトリー計測は、ＢＤ ＦＡＣＳＡｒｉａ（商標） ＩＩＩセルソーター（ＢＤ社製）を用いて行った。 After the antibody was added, the cells were incubated at 4 ° C. for 30 minutes. FACS buffer was added to make 50 mL, and the mixture was centrifuged (250 G × 10 minutes, 4 ° C.). After aspiration, the supernatant was suspended in 1 × 107 cells / 3 mL with FACS buffer, transferred to a 5 mL tube with a cell strainer (for flow cytometer, manufactured by Corning, USA, NY) and adjusted. Flow cytometry measurements were performed using a BD FACSAria ™ III cell sorter (manufactured by BD).
ＣＤ２０６陽性かつＣＸＣＲ４陽性の分画をＲｅｍａ細胞とし、ＣＤ２０６陽性かつＣＸＣＲ４陰性の分画をＮｏｎ－Ｒｅｍａ細胞としてそれぞれ同時にソートした。ソートの初期設定として、ＤＡＰＩ染色にて死細胞を除去し、目的細胞を含む領域の細胞集団（図１）をゲーティングした。ゲートされた領域の下層をダブレット除去し、アイソタイプ対照と単染色サンプルを用いて蛍光色素の漏れ込み調整を実施した。 The CD206-positive and CXCR4-positive fractions were simultaneously sorted as Rema cells, and the CD206-positive and CXCR4-negative fractions were simultaneously sorted as Non-Rema cells. As an initial setting for sorting, dead cells were removed by DAPI staining, and the cell population (FIG. 1) in the region containing the target cells was gated. The underlayer of the gated area was doublet-removed and a fluorochrome leak adjustment was performed using an isotype control and a single-stained sample.
実施例２ Ｒｅｍａ細胞の効果
（１）Ｒｅｍａ細胞の血管形成能
本実施例では、実施例１で単離したＲｅｍａ細胞の血管形成能を調べた。
Example 2 Effect of Rema cells (1) Angioplastic ability of Rema cells In this example, the angioplastic ability of Rema cells isolated in Example 1 was examined.
管腔形成能を持つヒト臍帯静脈内皮細胞（ＨＵＶＥＣ）（Ｌｏｎｚａ社、スイス、バーゼルより入手）を、８～１０継代で使用し、各群の細胞とＨＵＶＥＣと共培養した。９６ウェルのプレートに基底膜マトリックス（Ｃｏｒｎｉｎｇ（登録商標） Ｍａｔｒｉｇｅｌ（登録商標）（Ｃｏｒｎｉｎｇ社製，米国、ＮＹ））５０μｌ／ウェルを分注し、３７℃、３０分コーティングした。一方、実施例２で単離したＲｅＭａ細胞を、ＤｉＩ－Ａｃ－ＬＤＬで標識するために、ＩＭＤＭ ５００μｌ＋ＤｉＩ－Ａｃ－ＬＤＬ ５μｌに懸濁し、３７℃、１時間インキュベートした。比較例として、実施例１で調製したｎｏｎ－Ｒｅｍａ細胞を用いた。 Human umbilical vein endothelial cells (HUVEC) (obtained from Lonza, Basel, Switzerland) capable of forming a lumen were used in 8-10 passages and co-cultured with cells of each group and HUVEC. 50 μl / well of the basement membrane matrix (Corning® Matrix® (Corning, USA, NY)) was dispensed into a 96-well plate and coated at 37 ° C. for 30 minutes. On the other hand, the ReMa cells isolated in Example 2 were suspended in 500 μl of IMDM + 5 μl of DiI-Ac-LDL for labeling with DiI-Ac-LDL and incubated at 37 ° C. for 1 hour. As a comparative example, non-Rema cells prepared in Example 1 were used.
標識後、Ｒｅｍａ細胞又はｎｏｎ－Ｒｅｍａ細胞を１ｘ１０３／２５μｌ、及び、ＨＵＶＥＣを５ｘ１０３／２５μｌになるよう、各々ＰＢＳに再懸濁した。細胞数を調整した懸濁液を、Ｒｅｍａ細胞群とＨＵＶＥＣが１：１になるよう混和し、Ｍａｔｒｉｇｅｌ上に５０μｌずつ添加した。、３７℃、５％ＣＯ２下で、３～５時間培養した。培養後、ＩＸ８３蛍光顕微鏡（オリンパス社製）を用い、倍率１００倍で、形成した管腔数、並びに、管腔構造中に取り込まれている蛍光標識された細胞数（Ｒｅｍａ細胞又はｎｏｎ－Ｒｅｍａ細胞）を目視でカウントした。 After labeling, Rema cells or non-Rema cells were resuspended in PBS to 1x10 3/25 μl and HUVEC to 5x10 3/25 μl , respectively. The cell number-adjusted suspension was mixed so that the Rema cell group and HUVEC were 1: 1 and 50 μl each was added onto Matrix. , 37 ° C., 5% CO 2 for 3-5 hours. After culturing, using an IX83 fluorescence microscope (manufactured by Olympus) at a magnification of 100 times, the number of cavities formed and the number of fluorescently labeled cells (Rema cells or non-Rema cells) incorporated into the luminal structure. ) Was visually counted.
図２及び図３に結果を示す。図２は、ＨＵＶＥＣのみ（コントロール群）、Ｒｅｍａ細胞共培養、ｎｏｎ－Ｒｅｍａ細胞共培養の顕微鏡写真図である。図３Ａは、形成した管腔数、図３Ｂは管腔構造中に取り込まれている蛍光標識されたＲｅｍａ細胞およびｎｏｎ－Ｒｅｍａ細胞の細胞数の結果を示す。図３に示されるように、Ｒｅｍａ細胞は、コントロール群、ｎｏｎ－Ｒｅｍａ細胞と比較してＩｎ ｖｉｔｒｏ血管形成能が高いことが明らかになった。また、Ｒｅｍａ細胞は、ｎｏｎ－Ｒｅｍａ細胞よりも管腔構造中に取り込まれていることが明らかになった。 The results are shown in FIGS. 2 and 3. FIG. 2 is a micrograph of HUVEC only (control group), Rema cell co-culture, and non-Rema cell co-culture. FIG. 3A shows the number of lumens formed, and FIG. 3B shows the result of the number of fluorescently labeled Rema cells and non-Rema cells incorporated into the lumen structure. As shown in FIG. 3, it was revealed that the Rema cells have higher in vitro angioplasty ability than the control group, non-Rema cells. It was also revealed that Rema cells are more incorporated into the luminal structure than non-Rema cells.
（２）Ｒｅｍａ細胞の創傷治癒能
本実施例では、実施例１で取得されたＲｅｍａ細胞による創傷治癒能力を調べた。比較例として、ｎｏｎ－Ｒｅｍａ細胞（ＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（－））、を用いた。実験の概要を図４示す。
(2) Wound healing ability of Rema cells In this example, the wound healing ability of Rema cells obtained in Example 1 was investigated. As a comparative example, non-Rema cells (CD14 (+) CD206 (+) CXCR4 (-)) were used. The outline of the experiment is shown in FIG.
ヒト皮膚由来線維芽細胞を、培養培地（１０％ＦＢＳ含有ＤＭＥＭ）中で６～１０継代で使用したものを準備した。準備した線維芽細胞を、９６ウェルのＩｎｃｕＣｙｔｅ（登録商標）ＩｍａｇｅＬｏｃｋ Ｐｌａｔｅｓ（Ｅｓｓｅｎ ＢｉｏＳｃｉｅｎｃｅ社製、ａｎｎ Ａｒｂｏｒ，ＭＩ，米国）に０．８ｘ１０４細胞／ウェルの密度で播種した。ここに、Ｒｅｍａ細胞、ｎｏｎ－Ｒｅｍａ細胞又を、各々、０．８ｘ１０４細胞／ウェルの密度で、カルチャーインサート を用いて、線維芽細胞と接触しないように添加した。０．５％ＦＢＳ含有ＤＭＥＭ培地中で、３７℃、２４時間共培養した。 Human skin-derived fibroblasts were prepared for use in culture medium (DMEM containing 10% FBS) for 6 to 10 passages. Prepared fibroblasts were seeded in 96-well IncuCite® ImageLock Plates (Essen BioScience, Ann Arbor, MI, USA) at a density of 0.8x10 4 cells / well. Rema cells and non-Rema cells or non-Rema cells were added thereto at a density of 0.8x104 cells / well, respectively, using a culture insert so as not to come into contact with fibroblasts. Co-cultured in DMEM medium containing 0.5% FBS at 37 ° C. for 24 hours.
ＩｎｃｕＣｙｔｅ（登録商標）のスクラッチアッセイの９６ウェルのＷｏｕｎｄ Ｍａｋｅｒツールを用い、スクラッチ創傷を形成した。死細胞を除去するために、ＰＢＳで細胞を洗浄した。細胞共培養０、６、１２、２４時間後の写真を、全自動ハイスループット生細胞解析システムＩｎｃｕＣｙｔｅ（登録商標） Ｓ３により撮影した。結果を、図５に示す。さらに、ＩｎｃｕＣｙｔｅ（登録商標） 細胞遊走ソフトウェアにより創傷面積を算出した。０時間時を０とした場合の、６、１２、２４時間後の創傷の縮小率を図６に示す。
図６から明らかなようにＲｅｍａ細胞は、６、１２、２４時間後いずれにおいても、ｎｏｎ－Ｒｅｍａ細胞と比較して、高い創傷治癒能力を示した。
Scratch wounds were formed using the 96-well Wound Maker tool of the IncuCyte® scratch assay. Cells were washed with PBS to remove dead cells. Photographs after 0, 6, 12, and 24 hours of cell co-culture were taken by a fully automatic high-throughput live cell analysis system IncuCite® S3. The results are shown in FIG. In addition, wound area was calculated using IncuCyte® cell migration software. FIG. 6 shows the reduction rate of the wound after 6, 12, and 24 hours when 0 hour is set to 0.
As is clear from FIG. 6, Rema cells showed higher wound healing ability as compared with non-Rema cells at 6, 12, and 24 hours later.
実施例３ Ｒｅｍａ細胞を多く含む細胞群の培養
本実施例において、末梢血より単核球を単離し、Ｒｅｍａ細胞を多く含む細胞群の培養を行った。
Example 3 Culturing of a cell group containing a large amount of Rema cells In this example, mononuclear cells were isolated from peripheral blood, and a cell group containing a large amount of Rema cells was cultured.
（１）単核球の単離
糖尿病患者のボランティアから、ＢＤ バキュテイナ（登録商標）ＣＰＴ（商標）ＢＤ社製）単核球分離用採血管を用いて１００ｍＬの末梢血を採取した。採決方法、単核球の単離方法は実施例１（１）と同様である。
(1) Isolation of
（２）血清培地での培養
回収した単核球細胞を、１ｍＬの増殖培地に懸濁し、その一部を使用してトリパンブルーを用いた細胞数計数を行った。下記に示すいくつかの増殖培地で培養した。培地１は、５０ｎｇ／ｍｌ ＶＥＧＦ、１００ｎｇ／ｍｌ ＳＣＦ、１００ｎｇ／ｍｌ Ｆｌｔ－３リガンド、２０ｎｇ／ｍｌ ＴＰＯ、２０ｎｇ／ｍｌ及びＩＬ－６、１００単位／ｍＬの５種類の因子、並びに、ペニシリン、１００μｇ／ｍＬ ストレプトマイシン、０．５％ ＦＢＳを添加した ｓｔｅｍｌｉｎｅ（登録商標） ＩＩ Ｈｅｍａｔｏｐｏｉｅｔｉｃ Ｓｔｅｍ Ｃｅｌｌ Ｅｘｐａｎｓｉｏｎ Ｍｅｄｉｕｍ（シグマ－アルドリッチ社製、Ｃａｔ Ｎｏ．Ｓ０１９２を用いた。培地２は、５０ｎｇ／ｍｌ ＶＥＧＦ、１００ｎｇ／ｍｌ Ｆｌｔ－３リガンド、２０ｎｇ／ｍｌ ＴＰＯ、２０ｎｇ／の３種類の因子、並びに、ペニシリン、１００μｇ／ｍＬ ストレプトマイシン、０．５％ ＦＢＳを添加した ｓｔｅｍｌｉｎｅ（登録商標） ＩＩ Ｈｅｍａｔｏｐｏｉｅｔｉｃ Ｓｔｅｍ Ｃｅｌｌ Ｅｘｐａｎｓｉｏｎ Ｍｅｄｉｕｍ（シグマ－アルドリッチ社製、Ｃａｔ Ｎｏ．Ｓ０１９２を用いた。培地３は、５０ｎｇ／ｍｌ ＶＥＧＦ、１００ｎｇ／ｍｌ Ｆｌｔ－３リガンド、２０ｎｇ／ｍｌ ＴＰＯ、２０ｎｇ／の３種類の因子、並びに、ペニシリン、１００μｇ／ｍＬ ストレプトマイシン、０．５％ ＦＢＳ及び０．５％ ヒトアルブミン血清を添加した Ｉｓｃｏｖｅ’ｓ Ｍｏｄｉｆｉｅｄ Ｄｕｌｂｅｃｃｏ’ｓ Ｍｅｄｉｕｍ （ＩＭＤＭ）を用いた。
(2) Culture in serum medium The recovered mononuclear cells were suspended in 1 mL of growth medium, and a part of the cells was used to count the number of cells using trypan blue. The cells were cultured in several growth media shown below. Medium 1 contains 5 factors of 50 ng / ml VEGF, 100 ng / ml SCF, 100 ng / ml Flt-3 ligand, 20 ng / ml TPO, 20 ng / ml and IL-6, 100 units / mL, and penicillin, 100 μg. / ML streptomycin, 0.5% FBS-added stemline® II Hematopoietinic Stem Cell Expansion Medium (Cat No. S0192, manufactured by Sigma-Aldrich) was used. Medium 2 was 50 ng / ml VEGF, 100 ng / ml. Stemline® II Hematopoietin Stem Cell Expansion Medium with Penicillin, 100 μg / mL Streptomycin and 0.5% FBS, as well as 3 factors: Flt-3 ligand, 20 ng / ml TPO, 20 ng /. Cat No. S0192 manufactured by Cat No. S0192 was used. The medium 3 was 50 ng / ml VEGF, 100 ng / ml Flt-3 ligand, 20 ng / ml TPO, 20 ng /, and penicillin, 100 μg / mL streptomycin. Iscove's Modified Dulvecco's Medium (IMDM) supplemented with 0.5% FBS and 0.5% human albumin serum was used.
得られた細胞増殖培地中で、細胞濃度を１ｘ１０６／ｍＬに調製し、６穴培養プレートの各ウェルに２ｍＬずつ播種した。通常の培養条件（３７℃、５％ＣＯ２）で５日間培養した。５日間の培養終了後、遠心管に回収し、２５０ｘｇ（～１０００ｒｐｍ）で７－１０分間、遠心洗浄を３回実施した。遠心後、細胞数計測、生存率測定、ＱＣ（確認）試験用のサンプルを除き、残りの細胞はＰｌａｓｍａＬｙｔｅ Ａ、２．５％ ヒトアルブミン血清に懸濁し、８ｘ１０５細胞／ｍＬの濃度になるように調整した。 In the obtained cell proliferation medium, the cell concentration was adjusted to 1x10 6 / mL, and 2 mL was seeded in each well of the 6-well culture plate. The cells were cultured under normal culture conditions (37 ° C., 5% CO 2 ) for 5 days. After 5 days of culturing, the cells were collected in a centrifuge tube and centrifuged at 250 xg (~ 1000 rpm) for 7-10 minutes three times. After centrifugation, except for cell count, viability, and QC (confirmation) test samples, the remaining cells were suspended in PlasmaLyte A, 2.5% human albumin serum to a concentration of 8x10 5 cells / mL. Adjusted to.
（３）フローサイトメトリー解析
本実施例では、上記の培養によって得られた細胞群について、その中のＣＤ１４（＋）ＣＤ２０６（＋）ＣＸＣＲ４（＋）細胞の含有量を明らかにするため、フローサイトメトリー解析を行った。
(3) Flow cytometric analysis In this example, in order to clarify the content of CD14 (+) CD206 (+) CXCR4 (+) cells in the cell group obtained by the above culture, flow cytometry analysis is performed. Cytometric analysis was performed.
方法は実施例１と同様である。
それぞれの培地で培養した細胞群に含まれるＲｅｍａ細胞、Ｎｏｎ－Ｒｅｍａ細胞の割合を表１に示す。
The method is the same as in Example 1.
Table 1 shows the ratios of Rema cells and Non-Rema cells contained in the cell groups cultured in each medium.
実施例４ Ｒｅｍａ細胞を多く含む細胞群の効果
（１）Ｒｅｍａ細胞を多く含む細胞群の血管形成能
本実施例では、上記の培地２で取得されたＲｅｍａ細胞を多く含む細胞集団（Ｒｅｍａ細胞群）の血管形成能を調べた。血管形成能の試験方法は実施例２と同様である。
Example 4 Effect of cell group containing a large amount of Rema cells (1) Angiogenic ability of a cell group containing a large amount of Rema cells In this example, a cell population containing a large amount of Rema cells (Rema cell group) obtained in the above medium 2 ) Was investigated. The test method for angioplasty is the same as in Example 2.
図７及び図８に結果を示す。図７は、ＨＵＶＥＣ、Ｒｅｍａ細胞群共培養の顕微鏡写真図である。図８は、形成した管腔数の結果を示す。図８に示されるように、Ｒｅｍａ細胞群はコントロール群（ＨＵＶＥＣのみ）と比較してＩｎ ｖｉｔｒｏ血管形成能が高いことが明らかになった。 The results are shown in FIGS. 7 and 8. FIG. 7 is a micrograph of the HUVEC and Rema cell group co-culture. FIG. 8 shows the result of the number of lumens formed. As shown in FIG. 8, it was revealed that the Rema cell group had a higher in vitro angioplasty ability than the control group (HUVEC only).
（２）マウス下肢虚血モデルに対するＲｅｍａ細胞群の効果
本実施例では、実施例３で取得されたＲｅｍａ細胞を多く含む細胞集団（Ｒｅｍａ細胞群）のマウス下肢虚血モデルに対する効果を調べた。
(2) Effect of Rema cell group on mouse lower limb ischemia model In this example, the effect of the cell population (Rema cell group) containing a large amount of Rema cells obtained in Example 3 on the mouse lower limb ischemia model was investigated.
免疫不全マウスであるＢＡＬＢ／ｃＡＪｃｌ－ｎｕ／ｎｕヌードマウス（日本クレア より入手）用いて全身吸入麻酔下にて下肢虚血モデルを作製した。具体的には、全身吸入麻酔をした免疫不全マウスの鼡径部皮膚を切開し、鼠径部脂肪組織を除去した。切除部位直下の大腿動脈を６－０絹糸で結紮し、下流の伏在動脈をバイポーラーにて焼灼・切断した。 A lower limb ischemia model was prepared under systemic inhalation anesthesia using BALB / cAJcl-nu / nu nude mice (obtained from Claire Japan), which are immunodeficient mice. Specifically, the inguinal skin of immunodeficient mice subjected to systemic inhalation anesthesia was incised and the inguinal adipose tissue was removed. The femoral artery just below the excision site was ligated with 6-0 silk thread, and the downstream saphenous artery was cauterized and cut by bipolar.
実施例３の培地２で取得したＲｅｍａ細胞群を２，５００細胞／部位の濃度で虚血部位４ヶ所に筋肉内移植した。比較例として、生理食塩水を投与したグループを用いた。移植後に肉眼所見観察及びレーザードップラーによる血流測定を経時的に行った。観察終了後は組織免疫染色にて血管数、抗炎症マクロファージ数を計測し、細胞移植による組織再生効果を確認した。 The Rema cell group obtained in Medium 2 of Example 3 was intramuscularly transplanted to 4 ischemic sites at a concentration of 2,500 cells / site. As a comparative example, a group to which physiological saline was administered was used. After transplantation, macroscopic findings and blood flow measurement by laser Doppler were performed over time. After the observation was completed, the number of blood vessels and the number of anti-inflammatory macrophages were measured by tissue immunostaining, and the tissue regeneration effect by cell transplantation was confirmed.
本発明により、高い血管再生能及び創傷治癒能を有する新たな細胞もしくは細胞群が提供される。本発明の細胞もしくは細胞群は、例えば、難病の下肢虚血疾患の患者の治療薬としての使用できる。これにより、例えば、下肢切断の回避が可能になるなど、患者ＱＯＬの向上、介護負担の軽減などライフイノベーションをもたらすことが可能であり、社会的意義が大きい。また、新規な細胞もしくは細胞群の提供は、新たな治療戦略の構築につながるとともに、血管病変全般へに対する知見をもたしうる。 INDUSTRIAL APPLICABILITY The present invention provides a new cell or cell group having high angiogenic ability and wound healing ability. The cell or cell group of the present invention can be used, for example, as a therapeutic agent for a patient with an intractable lower limb ischemic disease. This makes it possible to bring about life innovation such as improvement of patient QOL and reduction of the burden of long-term care, such as avoidance of amputation of the lower limbs, and has great social significance. In addition, the provision of new cells or cell groups can lead to the construction of new therapeutic strategies and can provide insights into vascular lesions in general.
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