JP2014133703A - Composition for cell therapy for allotransplant including ssea-3 positive pluripotent stem cell that can be isolated from biological tissue - Google Patents

Composition for cell therapy for allotransplant including ssea-3 positive pluripotent stem cell that can be isolated from biological tissue Download PDF

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JP2014133703A
JP2014133703A JP2011076643A JP2011076643A JP2014133703A JP 2014133703 A JP2014133703 A JP 2014133703A JP 2011076643 A JP2011076643 A JP 2011076643A JP 2011076643 A JP2011076643 A JP 2011076643A JP 2014133703 A JP2014133703 A JP 2014133703A
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Mari Idesawa
真理 出澤
Masayori Yoshida
正順 吉田
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Abstract

PROBLEM TO BE SOLVED: To provide a composition for cell therapy for allotransplant including a pluripotent stem cell.SOLUTION: A composition for cell therapy for allotransplant includes a SSEA-3 positive pluripotent stem cell that can be isolated from biological tissue.

Description

本発明は、生体組織由来のSSEA-3陽性の多能性幹細胞を含む他家移植用細胞治療用組成物に関する。   The present invention relates to a composition for cell therapy for xenotransplantation comprising SSEA-3 positive pluripotent stem cells derived from living tissue.

近年、組織再生に貢献し得る成人幹細胞又は組織幹細胞が注目されている。
成体から得られる分化能を有する細胞として、例えば骨、軟骨、脂肪細胞、神経細胞、骨格筋等への分化能を有する骨髄間葉系細胞画分(MSC: Bone marrow stromal cell)が報告されている(非特許文献1及び2を参照)。しかしながら、骨髄間葉系細胞画分は様々な細胞を含む細胞群であり、その分化能は多様でありながら本体がはっきりせず、また特定の細胞に分化させるために特定の化合物による刺激や遺伝子導入等が必要であり、分化誘導システムを構築する必要があった。
In recent years, adult stem cells or tissue stem cells that can contribute to tissue regeneration have attracted attention.
As a cell having differentiation potential obtained from an adult, for example, a bone marrow mesenchymal cell fraction (MSC: Bone marrow stromal cell) having differentiation potential into bone, cartilage, adipocyte, nerve cell, skeletal muscle, etc. has been reported. (See Non-Patent Documents 1 and 2). However, the bone marrow mesenchymal cell fraction is a group of cells containing various cells, and its differentiation ability is diverse, but the body is not clear, and in order to differentiate into specific cells, stimulation with specific compounds and genes It was necessary to introduce a differentiation induction system.

さらに、成体由来の多能性幹細胞としてiPS細胞(induced pluripotent stem cell)(特許文献1、特許文献2、非特許文献3等を参照)が報告されていた。しかしながら、iPS細胞の樹立には、間葉系細胞である皮膚線維芽細胞画分(dermal fibroblast)に特定の遺伝子や特定の化合物を体細胞に導入するという特定の物質を用いた誘導操作が必要であった。   Furthermore, iPS cells (induced pluripotent stem cells) (see Patent Document 1, Patent Document 2, Non-Patent Document 3, etc.) have been reported as adult-derived pluripotent stem cells. However, in order to establish iPS cells, an induction procedure using a specific substance that introduces a specific gene or a specific compound into a somatic cell into the dermal fibroblast fraction, which is a mesenchymal cell, is necessary. Met.

また、生体組織から単離できるSSEA-3陽性の多能性幹細胞であって、それまで知られていなかった生体由来の幹細胞についての報告があった(特許文献3及び非特許文献4等を参照)。   In addition, SSEA-3-positive pluripotent stem cells that can be isolated from living tissues, and there have been reports on stem cells derived from living bodies that have not been known so far (see Patent Document 3 and Non-Patent Document 4). ).

特許第4183742号公報Japanese Patent No.4183742 特開2008-307007号公報JP 2008-307007 A 国際公開第WO2011/007900号国際公開パンフレットInternational Publication No.WO2011 / 007900 International Publication Pamphlet

M. DEZAWA et al., The Journal of Clinical Investigation, 113, 12, pp. 1701-1710, (2004)M. DEZAWA et al., The Journal of Clinical Investigation, 113, 12, pp. 1701-1710, (2004) M. DEZAWA et al., SCIENCE, 2005 July 8, 309, pp. 314-317, (2005)M. DEZAWA et al., SCIENCE, 2005 July 8, 309, pp. 314-317, (2005) Okita K. et al. SCIENCE, 2008 Nov 7, 322(5903), pp.949-953Okita K. et al. SCIENCE, 2008 Nov 7, 322 (5903), pp.949-953 Proc.Natl.Acad.Sci USA, 107(19):8639-43, 2010Proc.Natl.Acad.Sci USA, 107 (19): 8639-43, 2010

本発明は、SSEA-3陽性であり、HLA class II抗原を発現しない、多能性幹細胞を含む他家移植用細胞治療用組成物の提供を目的とする。   An object of the present invention is to provide a composition for cell therapy for xenotransplantation containing pluripotent stem cells that are positive for SSEA-3 and do not express HLA class II antigen.

本発明者らは、皮膚線維芽細胞及び骨髄細胞よりSSEA-3陽性であり、従来の幹細胞には認められない抗原発現パターンを有する幹細胞を単離し、Muse(Multilineage-differentiating Stress Enduring cells)細胞と名付けた(国際公開第WO2011/007900号国際公開パンフレット、Proc.Natl.Acad.Sci USA, 107(19):8639-43, 2010)。 The present inventors are SSEA-3 positive than skin fibroblasts and bone marrow cells, stem cells having a conventional trunk antigen expression patterns not found in cells isolated, Muse (Mu ltilineage-differentiating S tress E nduring cells ) Cells (International Publication No. WO2011 / 007900, International Publication Pamphlet, Proc. Natl. Acad. Sci USA, 107 (19): 8639-43, 2010).

本発明者等は、得られたMuse細胞の特性を解析した結果、該細胞がHLA抗原のうち、class I抗原をは発現しているが、class II抗原は発現していないことを見出し、他家移植した場合に免疫抑制剤を併用しなくても、拒絶されない可能性があると考え鋭意検討を行った。   As a result of analyzing the characteristics of the obtained Muse cells, the present inventors have found that the cells express class I antigen but not class II antigen among HLA antigens. Even if an immunosuppressant was not used in combination with home transplantation, it was considered that it may not be rejected, and an intensive study was conducted.

その結果、Muse細胞を他家移植に用い得ることを見出し、Muse細胞を含む他家移植用細胞治療用組成物である本発明を完成させるに至った。   As a result, it has been found that Muse cells can be used for allogeneic transplantation, and the present invention which is a composition for cell therapy for allogeneic transplantation containing Muse cells has been completed.

すなわち、本発明は以下の通りである。
[1] 生体組織から単離できるSSEA-3陽性であって、HLA class II抗原を発現しない多能性幹細胞を含む、他家移植用細胞治療用組成物。
[2] さらに、多能性幹細胞がCD105陽性である、[1]の他家移植用細胞治療用組成物。
[3] さらに、多能性幹細胞がCD117陰性及びCD146陰性である、[1]又は[2]の他家移植用細胞治療用組成物。
[4] さらに、多能性幹細胞がCD117陰性、CD146陰性、NG2陰性、CD34陰性、vWF陰性及びCD271陰性である、[1]又は[2]の他家移植用細胞治療用組成物。
[5] さらに、多能性幹細胞がCD34陰性、CD117陰性、CD146陰性、CD271陰性、NG2陰性、vWF陰性、Sox10陰性、Snail陰性、Slug陰性、Tyrp1陰性及びDct陰性である、[1]又は[2]の他家移植用細胞治療用組成物。
[6] さらに、多能性幹細胞がテロメラーゼ活性が低いか又は無い、[1]〜[5]のいずれかの他家移植用細胞治療用組成物。
[7] さらに、多能性幹細胞が三胚葉に分化する能力を持つ、[1]〜[6]のいずれかの他家移植用細胞治療用組成物。
[8] さらに、多能性幹細胞が腫瘍性増殖を示さない、[1]〜[7]のいずれかの他家移植用細胞治療用組成物。
[9] さらに、多能性幹細胞がセルフリニューアル能を持つ、[1]〜[8]のいずれかの他家移植用細胞治療用組成物。
[10] さらに、多能性幹細胞がストレス耐性である、[1]〜[9]のいずれかの他家移植用細胞治療用組成物。
[11] さらに、多能性幹細胞が貪食能が高い、[1]〜[10]のいずれかの他家移植用細胞治療用組成物。
[12] さらに、多能性幹細胞が以下に示す22個のオドラント受容体の少なくとも一つが陽性の[1]〜[11]のいずれかの他家移植用細胞治療用組成物:
olfactory receptor, family 8, subfamily G, member 2 (OR8G2);
olfactory receptor, family 7, subfamily G, member 3 (OR7G3);
olfactory receptor, family 4, subfamily D, member 5 (OR4D5);
olfactory receptor, family 5, subfamily AP, member 2 (OR5AP2);
olfactory receptor, family 10, subfamily H, member 4 (OR10H4);
olfactory receptor, family 10, subfamily T, member 2 (OR10T2);
olfactory receptor, family 2, subfamily M, member 2 (OR2M2);
olfactory receptor, family 2, subfamily T, member 5 (OR2T5);
olfactory receptor, family 7, subfamily D, member 4 (OR7D4);
olfactory receptor, family 1, subfamily L, member 3 (OR1L3);
olfactory receptor, family 4, subfamily N, member 4 (OR4N4);
olfactory receptor, family 2, subfamily A, member 7 (OR2A7);
guanine nucleotide binding protein (G protein), alpha activating activity polypeptide, olfactory type (GNAL);
olfactory receptor, family 6, subfamily A, member 2 (OR6A2);
olfactory receptor, family 2, subfamily B, member 6 (OR2B6);
olfactory receptor, family 2, subfamily C, member 1 (OR2C1);
olfactory receptor, family 52, subfamily A, member 1 (OR52A1);
olfactory receptor, family 10, subfamily H, member 3 (OR10H3);
olfactory receptor, family 10, subfamily H, member 2 (OR10H2);
olfactory receptor, family 51, subfamily E, member 2 (OR51E2);
olfactory receptor, family 5, subfamily P, member 2 (OR5P2);及び
olfactory receptor, family 10, subfamily P, member 1 (OR10P1)。
[13] さらに、多能性幹細胞が以下に示す5個のケモカイン受容体の少なくとも一つが陽性の[1]〜[12]のいずれかの他家移植用細胞治療用組成物:
chemokine (C-C motif) receptor 5(CCR5);
chemokine (C-X-C motif) receptor 4(CXCR4);
chemokine (C-C motif) receptor 1(CCR1);
Duffy blood group, chemokine receptor(DARC);及び
chemokine (C-X-C motif) receptor 7(CXCR7)。
[14] さらに、多能性幹細胞が中胚葉系組織または間葉系組織由来である、[1]〜[13]のいずれかの他家移植用細胞治療用組成物。
That is, the present invention is as follows.
[1] A composition for cell therapy for allogeneic transplantation, comprising pluripotent stem cells that are SSEA-3 positive and that do not express HLA class II antigen, which can be isolated from a living tissue.
[2] Furthermore, the composition for cell therapy for autologous transplantation, wherein the pluripotent stem cells are CD105 positive.
[3] The composition for cell therapy for allogeneic transplantation according to [1] or [2], wherein the pluripotent stem cells are CD117 negative and CD146 negative.
[4] The composition for cell therapy for allogeneic transplantation according to [1] or [2], wherein the pluripotent stem cells are CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative and CD271 negative.
[5] Furthermore, the pluripotent stem cells are CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snail negative, Slug negative, Tyrp1 negative and Dct negative. [1] or [ 2] A composition for cell therapy for allogeneic transplantation.
[6] The composition for cell therapy for allogeneic transplantation according to any one of [1] to [5], wherein the pluripotent stem cell has low or no telomerase activity.
[7] The composition for cell therapy for allotransplantation according to any one of [1] to [6], further having the ability of pluripotent stem cells to differentiate into three germ layers.
[8] The composition for cell therapy for allotransplantation according to any one of [1] to [7], wherein the pluripotent stem cells do not exhibit neoplastic growth.
[9] The composition for cell therapy for allogeneic transplantation according to any one of [1] to [8], wherein the pluripotent stem cells further have a self-renewal ability.
[10] The composition for cell therapy for allogeneic transplantation according to any one of [1] to [9], wherein the pluripotent stem cells are stress resistant.
[11] The composition for cell therapy for allogeneic transplantation according to any one of [1] to [10], wherein the pluripotent stem cells have high phagocytic ability.
[12] The composition for cell transplantation for transplantation according to any one of [1] to [11], wherein the pluripotent stem cell is positive for at least one of the 22 odorant receptors shown below:
olfactory receptor, family 8, subfamily G, member 2 (OR8G2);
olfactory receptor, family 7, subfamily G, member 3 (OR7G3);
olfactory receptor, family 4, subfamily D, member 5 (OR4D5);
olfactory receptor, family 5, subfamily AP, member 2 (OR5AP2);
olfactory receptor, family 10, subfamily H, member 4 (OR10H4);
olfactory receptor, family 10, subfamily T, member 2 (OR10T2);
olfactory receptor, family 2, subfamily M, member 2 (OR2M2);
olfactory receptor, family 2, subfamily T, member 5 (OR2T5);
olfactory receptor, family 7, subfamily D, member 4 (OR7D4);
olfactory receptor, family 1, subfamily L, member 3 (OR1L3);
olfactory receptor, family 4, subfamily N, member 4 (OR4N4);
olfactory receptor, family 2, subfamily A, member 7 (OR2A7);
guanine nucleotide binding protein (G protein), alpha activating activity polypeptide, olfactory type (GNAL);
olfactory receptor, family 6, subfamily A, member 2 (OR6A2);
olfactory receptor, family 2, subfamily B, member 6 (OR2B6);
olfactory receptor, family 2, subfamily C, member 1 (OR2C1);
olfactory receptor, family 52, subfamily A, member 1 (OR52A1);
olfactory receptor, family 10, subfamily H, member 3 (OR10H3);
olfactory receptor, family 10, subfamily H, member 2 (OR10H2);
olfactory receptor, family 51, subfamily E, member 2 (OR51E2);
olfactory receptor, family 5, subfamily P, member 2 (OR5P2); and
olfactory receptor, family 10, subfamily P, member 1 (OR10P1).
[13] Furthermore, the composition for cell therapy for allogeneic transplantation according to any one of [1] to [12], wherein the pluripotent stem cell is positive for at least one of the five chemokine receptors shown below:
chemokine (CC motif) receptor 5 (CCR5);
chemokine (CXC motif) receptor 4 (CXCR4);
chemokine (CC motif) receptor 1 (CCR1);
Duffy blood group, chemokine receptor (DARC); and
chemokine (CXC motif) receptor 7 (CXCR7).
[14] The composition for cell therapy for allogeneic transplantation according to any one of [1] to [13], wherein the pluripotent stem cells are derived from mesodermal or mesenchymal tissue.

生体組織より得られたSSEA-3陽性であり、従来の幹細胞には認められない抗原発現パターンを有する多能性幹細胞であるMuse細胞は、HLA class II抗原を発現しない。そのため、細胞を他家移植しても拒絶されることがない。従って、Muse細胞を含む他家移植用細胞治療用組成物は、再生医療のための細胞治療に用いることができる。   Muse cells, which are pluripotent stem cells that are SSEA-3 positive and obtained from living tissue and have an antigen expression pattern not found in conventional stem cells, do not express HLA class II antigen. Therefore, even if the cells are transplanted to another family, they will not be rejected. Therefore, the cell therapy composition for transplantation containing Muse cells can be used for cell therapy for regenerative medicine.

ヒト骨髄間葉系細胞由来のSSEA-3陽性細胞のHLA class I及びHLA class II抗原の発現を示す図である。It is a figure which shows the expression of the HLA class I and HLA class II antigen of the SSEA-3 positive cell derived from a human bone marrow mesenchymal cell. ヒト線維芽細胞由来のSSEA-3陽性細胞のHLA class I及びHLA class II抗原の発現を示す図である。It is a figure which shows the expression of the HLA class I and HLA class II antigen of the SSEA-3 positive cell derived from a human fibroblast. Muse細胞及び非Muse細胞におけるHLA Class Iの発現を示す図である。It is a figure which shows the expression of HLA Class I in a Muse cell and a non-Muse cell. Muse細胞及び非Muse細胞におけるHLA Class IIの発現を示す図である。It is a figure which shows the expression of HLA Class II in a Muse cell and a non-Muse cell. Muse細胞及び非Muse細胞におけるAlexa568との反応性を示す図である。It is a figure which shows the reactivity with Alexa568 in a Muse cell and a non-Muse cell.

以下、本発明を詳細に説明する。
本発明は多能性幹細胞を含む他家移植のための細胞治療用組成物又は細胞治療剤である。
Hereinafter, the present invention will be described in detail.
The present invention is a cell therapy composition or cell therapy agent for allogeneic transplantation containing pluripotent stem cells.

Muse細胞は、Muse細胞と呼ばれる多能性幹細胞である。本発明において、Muse細胞という場合、細胞画分も含み、Muse細胞画分はMuse細胞を少なくとも一定量含む細胞群のことをいう。例えば、Muse細胞画分は、Muse細胞を1%以上、10%以上、30%以上、50%以上、70%以上、90%以上、又は95%以上含む細胞群であり、Muse細胞の培養によって得られる細胞塊やMuse細胞を濃縮した細胞群を含む。また、前記Muse細胞画分を実質的に均一なMuse細胞画分ということもある。   Muse cells are pluripotent stem cells called Muse cells. In the present invention, the term “Muse cell” includes a cell fraction, and the Muse cell fraction refers to a cell group containing at least a certain amount of Muse cells. For example, the Muse cell fraction is a group of cells containing 1% or more, 10% or more, 30% or more, 50% or more, 70% or more, 90% or more, or 95% or more of Muse cells. Including cell clusters obtained by concentrating the resulting cell mass and Muse cells. The Muse cell fraction may be referred to as a substantially uniform Muse cell fraction.

Muse細胞は、生体組織から単離できるSSEA-3陽性の多能性幹細胞である。
生体は、哺乳動物の生体をいい、ある程度発生が進んだ動物体をいう。本発明において、生体には、受精卵や胞胚期より発生段階が前の胚は含まれないが、胎児や胞胚を含む胞胚期以降の発生段階の胚は含まれる。哺乳動物は限定されないが、例えばヒト、サル等の霊長類、マウス、ラット、ウサギ、モルモット等のげっ歯類、ネコ、イヌ、ヒツジ、ブタ、ウシ、ウマ、ロバ、ヤギ、フェレット等が含まれる。Muse細胞は、生体の組織由来である点で、胚性幹細胞(ES細胞)や胚性生殖幹細胞(EG細胞)と明確に区別される。
Muse cells are SSEA-3 positive pluripotent stem cells that can be isolated from living tissues.
A living body refers to a living body of a mammal, and refers to an animal body that has developed to some extent. In the present invention, the living body does not include embryos whose developmental stage is earlier than the fertilized egg or blastocyst stage, but includes embryos in the developmental stage after the blastocyst stage including the fetus and blastocyst. Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits, guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, ferrets, etc. . Muse cells are clearly distinguished from embryonic stem cells (ES cells) and embryonic germ stem cells (EG cells) in that they are derived from living tissue.

中胚葉系組織とは、動物の初期発生途上で現れる中胚葉起源の組織をいい、筋肉系組織、結合組織、循環系組織、排泄系組織、生殖系組織等が含まれる。例えば、Muse細胞は、骨髄液や真皮結合組織等の皮膚組織から得ることができる。   Mesodermal tissue refers to tissue of mesoderm origin that appears in the early development of animals, and includes muscular tissue, connective tissue, circulatory tissue, excretory tissue, reproductive tissue, and the like. For example, Muse cells can be obtained from skin tissues such as bone marrow fluid and dermal connective tissue.

間葉系組織とは、骨、軟骨、脂肪、血液、骨髄、骨格筋、真皮、靭帯、腱、心臓、などの組織をいう。例えば、Muse細胞は、骨髄や皮膚から得ることができる。また、臍帯や脂肪幹細胞から得ることもできる。   Mesenchymal tissue refers to tissues such as bone, cartilage, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, and heart. For example, Muse cells can be obtained from bone marrow or skin. It can also be obtained from umbilical cords or adipose stem cells.

細胞が組織から直接得ることができるとは、組織から単離することができ、外来遺伝子や外来タンパク質の導入又は化合物の投与などの化合物処理等の人為的な誘導操作を経ずに得られることを意味する。ここで、外来遺伝子は、限定されないが、例えば体細胞の核を初期化し得る遺伝子をいい、例えば、Oct3/4遺伝子等のOctファミリー遺伝子、Klf遺伝子等のKlfファミリー遺伝子、c-Myc遺伝子等のMycファミリー遺伝子、Sox2遺伝子等のSoxファミリー遺伝子が挙げられる。また、外来タンパク質としてはこれらの遺伝子がコードするタンパク質やサイトカインが挙げられる。さらに、化合物としては、例えば、上記の体細胞の核を初期化し得る遺伝子の発現を誘導する低分子化合物やDMSO、還元剤として機能する化合物, DNA メチル化剤等が挙げられる。Muse細胞は、生体あるいは組織から直接得ることができるという点で、iPS(induced pluripotent stem cell)細胞及びES細胞とは明確に区別される。なお、本発明においては、細胞の培養、細胞の表面マーカーを指標に細胞又は細胞画分を単離すること、細胞を細胞ストレスに曝露すること、及び細胞に物理的衝撃を与えることは、人為的な誘導操作には含まれない。また、Muse細胞は、リプログラミング又は脱分化の誘導を必要とせずに得られることを特徴としてもよい。   That cells can be obtained directly from tissue means that they can be isolated from tissue and obtained without artificial induction procedures such as compound treatment such as introduction of foreign genes or proteins, or administration of compounds. Means. Here, the foreign gene is not limited, but refers to a gene that can initialize the nucleus of a somatic cell, for example, an Oct family gene such as an Oct3 / 4 gene, a Klf family gene such as a Klf gene, a c-Myc gene, etc. Examples include Sox family genes such as Myc family gene and Sox2 gene. Examples of foreign proteins include proteins and cytokines encoded by these genes. Furthermore, examples of the compound include a low molecular weight compound that induces expression of a gene that can reprogram the somatic cell nucleus, DMSO, a compound that functions as a reducing agent, a DNA methylating agent, and the like. Muse cells are clearly distinguished from iPS (induced pluripotent stem cell) cells and ES cells in that they can be obtained directly from a living body or tissue. In the present invention, cell culture, isolation of cells or cell fractions using cell surface markers as indicators, exposure of cells to cell stress, and physical impact on cells It is not included in typical guidance operations. Further, the Muse cell may be obtained without requiring reprogramming or induction of dedifferentiation.

Muse細胞は、生体の中胚葉系組織又は間葉系組織等に存在していると考えられ、本発明においては、これらの組織に存在している細胞又は細胞画分を単離する。Muse細胞は、例えば、骨髄に存在しており、骨髄から血液等を介して生体の各組織に供給される可能性がある。このため骨髄や、皮膚等の生体の各組織、さらには血液から単離することが可能である。   Muse cells are considered to be present in living mesodermal or mesenchymal tissues, and in the present invention, cells or cell fractions present in these tissues are isolated. Muse cells are present, for example, in the bone marrow, and may be supplied from the bone marrow to each tissue of the living body via blood or the like. For this reason, it can be isolated from bone marrow, living body tissues such as skin, and blood.

多能性幹細胞とは、pluripotencyを有している細胞をいい、以下の特性を有する。
(1) Nanog、Oct3/4、SSEA-3、PAR-4及びSox2等の多能性マーカー(Pluripotent marker)を発現する。
(2) 1細胞から増殖し、自己のクローンを作り続けるクローナリティー(Clonality)を有する。
(3) 自己複製(セルフリニューアル)能を有する。
(4) 3胚葉系(内胚葉系、中胚葉系及び外胚葉系)へin vitro及びin vivoで分化し得る。
(5) マウスの精巣や皮下に移植した場合、3胚葉系への分化を呈する。
(6) アルカリフォスファターゼ染色で陽性となる。
A pluripotent stem cell refers to a cell having pluripotency and has the following characteristics.
(1) Express pluripotent markers such as Nanog, Oct3 / 4, SSEA-3, PAR-4 and Sox2.
(2) It has the clonality that grows from one cell and keeps making its own clone.
(3) Has the ability to self-replicate (self-renewal)
(4) It can differentiate into three germ layers (endoderm, mesodermal and ectoderm) in vitro and in vivo.
(5) When transplanted into the mouse testis or subcutaneously, it exhibits differentiation into three germ layers.
(6) Positive by alkaline phosphatase staining.

Muse細胞は、pluripotencyを有している点で、成人幹細胞、組織幹細胞とは明確に区別される。また、Muse細胞は、pluripotencyを有している単一の又は複数の細胞として単離されている点で、骨髄間葉系細胞等の細胞画分とは明確に区別される。   Muse cells are clearly distinguished from adult stem cells and tissue stem cells in that they have pluripotency. In addition, Muse cells are clearly distinguished from cell fractions such as bone marrow mesenchymal cells in that they are isolated as single or multiple cells having pluripotency.

さらに、Muse細胞は、以下の特性を有する。
(i) 増殖速度が比較的緩やかで、分裂周期が1日以上、例えば1.2〜1.5日である。ただし、ES細胞やiPS細胞が示すような無限増殖は示さない。
(ii) 免疫不全マウスに移植した場合に内胚葉系、中胚葉系及び外胚葉系への分化を示す。ES細胞やiPS細胞ではテラトーマが短期間で癌化するのに比べ、半年以上癌化しないことを特徴とする。
(iii) 浮遊培養により胚様体様細胞塊を形成する。
(iv) 浮遊培養にて胚様体様細胞塊を形成し、10日程度で増殖が停止する。その後、接着培養に移動させることにより再増殖する。
(v) 増殖の際に非対称分裂を伴う。
(vi) 核型は正常である。
(vii) テロメラーゼ活性が無いか又は低い。ここで、テロメラーゼ活性が無いか又は低いとは、例えばTRAPEZE XL telomerase detection kit(Millipore社)を用いてテロメラーゼ活性を検出した場合に検出できないか又は低いことをいう。テロメラーゼ活性が低いとは、例えば、ヒト線維芽細胞と同程度のテロメラーゼ活性を有しているか、あるいはHela細胞に比べて1/5以下、好ましくは1/10以下のテロメラーゼ活性を有していることをいう。
(viii) メチル化の状態については、Muse細胞から誘導したiPS細胞に関してはNanogおよびOct3/4のプロモータ領域のメチル化レベルが低い。
(ix) 貪食能が高い。
(x) 腫瘍性増殖を示さない。ここで、腫瘍性増殖を示さないとは、浮遊培養を行った場合、一定の大きさの細胞塊(クラスター)に達すると増殖が止まり、無限増殖しないことをいう。また免疫不全マウスの精巣に移植しても奇形腫を形成しないことである。なお、上記(i)〜(iv)等も腫瘍性増殖を示さないことに関連する。
Furthermore, Muse cells have the following characteristics.
(I) The growth rate is relatively slow and the division cycle is 1 day or more, for example, 1.2 to 1.5 days. However, it does not show infinite proliferation as shown by ES cells and iPS cells.
(Ii) shows differentiation into endoderm, mesoderm and ectoderm when transplanted into immunodeficient mice. In ES cells and iPS cells, teratoma does not become cancerous for more than half a year compared to canceration in a short period of time.
(Iii) An embryoid body-like cell mass is formed by suspension culture.
(Iv) An embryoid body-like cell mass is formed in suspension culture, and the growth stops in about 10 days. Thereafter, it is re-growth by moving to adherent culture.
(V) Accompanying asymmetric division during growth.
(Vi) The karyotype is normal.
(Vii) No or low telomerase activity. Here, the absence or low telomerase activity means that it cannot be detected or is low when telomerase activity is detected using, for example, TRAPEZE XL telomerase detection kit (Millipore). Low telomerase activity means, for example, telomerase activity comparable to that of human fibroblasts, or telomerase activity of 1/5 or less, preferably 1/10 or less, compared to Hela cells. That means.
(Viii) Regarding the methylation status, the methylation levels of the Nanog and Oct3 / 4 promoter regions are low for iPS cells derived from Muse cells.
(Ix) High craving ability.
(X) Does not show neoplastic growth. Here, not exhibiting neoplastic growth means that when suspension culture is performed, the growth stops when the cell mass (cluster) of a certain size is reached, and does not grow infinitely. Also, teratomas do not form when transplanted into the testis of immunodeficient mice. The above (i) to (iv) are also related to not showing neoplastic growth.

すなわち、本発明の細胞は、例えば以下の多能性幹細胞である。
(A) 生体の中胚葉系組織又は間葉系組織等から得られる細胞であって、当該細胞内に化学物質、外来遺伝子又は外来タンパク質を導入することなく直接得ることができる多能性幹細胞。
(B) 生体の中胚葉系組織又は間葉系組織等が、骨髄、皮膚、血液、臍帯、脂肪などからなる群から選択される上記(1)の特性を有する多能性幹細胞。
(C) リプログラミングまたは脱分化を誘導することなく得ることができる、上記(A)又は(B)の多能性幹細胞。
(D) 精巣へ移植した場合に、少なくとも半年間は癌化しない、上記(A)又は(B)の多能性幹細胞。
(E) ES細胞、iPS細胞のように無限増殖を示さない、上記(A)又は(B)の多能性幹細胞。
(F) 生体の中胚葉系組織又は間葉系組織等由来の多能性幹細胞であって、生体の中胚葉系組織又は間葉系組織等の細胞をプロテアーゼで処理したときに生き残る、プロテアーゼに耐性である多能性幹細胞。
That is, the cell of the present invention is, for example, the following pluripotent stem cell.
(A) A pluripotent stem cell obtained from a mesodermal or mesenchymal tissue of a living body, which can be obtained directly without introducing a chemical substance, a foreign gene or a foreign protein into the cell.
(B) A pluripotent stem cell having the characteristic of (1) above, wherein the mesodermal or mesenchymal tissue of a living body is selected from the group consisting of bone marrow, skin, blood, umbilical cord, fat and the like.
(C) The pluripotent stem cell according to (A) or (B), which can be obtained without inducing reprogramming or dedifferentiation.
(D) The pluripotent stem cell according to (A) or (B), which does not become cancerous for at least half a year when transplanted to the testis.
(E) The pluripotent stem cell according to (A) or (B), which does not exhibit infinite proliferation like ES cells and iPS cells.
(F) A pluripotent stem cell derived from a mesodermal or mesenchymal tissue in a living body, which survives when cells such as a mesodermal or mesenchymal tissue in a living body are treated with a protease. Pluripotent stem cells that are resistant.

Muse細胞は、Muse細胞の表面に多く発現している細胞表面マーカーを利用して単離することができ、例えばSSEA-3の発現を指標に単離することができる。Muse細胞をSSEA-3陽性Muse細胞ということもある。さらに、Muse細胞は多能性幹細胞マーカーであるCD105を発現しており、SSEA-3陽性であり、CD105陽性である。従って、SSEA-3及びCD105の両方の発現を指標に単離することができる。これらの細胞表面マーカーを利用することにより、Muse細胞を単一細胞として単離でき、単離した単一細胞を、培養により増殖させることができる。なお、本発明は、ヒト以外の哺乳動物の生体組織からSSEA-3に相当するマーカーによって単離できる多能性幹細胞をも含むものとする。   Muse cells can be isolated using cell surface markers that are highly expressed on the surface of Muse cells. For example, SSEA-3 expression can be used as an indicator. Muse cells are sometimes referred to as SSEA-3 positive Muse cells. Furthermore, Muse cells express CD105, a pluripotent stem cell marker, are SSEA-3 positive and CD105 positive. Therefore, the expression of both SSEA-3 and CD105 can be isolated using as an index. By using these cell surface markers, Muse cells can be isolated as single cells, and the isolated single cells can be grown in culture. The present invention also includes pluripotent stem cells that can be isolated from living tissues of mammals other than humans with a marker corresponding to SSEA-3.

一方、Muse細胞は、NG2、CD34、vWF(フォンビルブランド因子)、c-kit(CD117)、CD146、CD271(NGFR)が陰性である。さらに、Sox10、Snai1、Slug、Tyrp1、Dctが陰性である。   On the other hand, Muse cells are negative for NG2, CD34, vWF (von Willebrand factor), c-kit (CD117), CD146, and CD271 (NGFR). Furthermore, Sox10, Snai1, Slug, Tyrp1, and Dct are negative.

NG2、CD34、vWF、CD117、CD146、CD271などの表面抗原が陰性かどうか、発現が弱いかどうかはこれらの抗原に対する抗体であって、発色酵素、蛍光化合物等で標識した抗体を用いて細胞が染色されたか否かを顕微鏡観察等により決定することができる。例えば、これらの抗体を用いて細胞を免疫染色して、表面抗原の有無を決定することができ、また該抗体を結合させた磁性ビーズを用いても決定することができる。また、FACS又はフローサイトメーターを用いても表面抗原があるかどうか決定することができる。フローサイトメーターとしては例えばFACSAria(ベクトン・ディッキンソン社製)、FACS vantage(ベクトン・ディッキンソン社製)、FACS Calibur(ベクトン・ディッキンソン社製)等を用いることができる。   Whether surface antigens such as NG2, CD34, vWF, CD117, CD146, and CD271 are negative or weakly expressed is an antibody against these antigens, and cells are labeled with antibodies labeled with chromogenic enzymes, fluorescent compounds, etc. Whether it is stained or not can be determined by microscopic observation or the like. For example, cells can be immunostained using these antibodies to determine the presence or absence of surface antigens, or can be determined using magnetic beads to which the antibodies are bound. It can also be determined whether there is a surface antigen using a FACS or flow cytometer. As the flow cytometer, for example, FACSAria (Becton Dickinson), FACS vantage (Becton Dickinson), FACS Calibur (Becton Dickinson), etc. can be used.

また、Sox10、Snai1、Slug、Tyrp1、Dctなどの転写因子に関してはRT-PCR等の手法により発現を調べることもできる。   In addition, expression of transcription factors such as Sox10, Snai1, Slug, Tyrp1, and Dct can be examined by a technique such as RT-PCR.

これらの表面抗原が陰性とは、上記のようにFACSを用いて分析した場合に、陽性細胞としてソーティングされないこと、あるいはRT-PCRにより発現を調べた場合に、発現が認められないことをいい、これらの手法により検出できない程度発現していたとしても、本発明においては陰性とする。また、上記マーカーが陽性であることが公知の造血幹細胞等の細胞と同時に測定を行い、これらの陽性細胞と比較して、ほとんど検出されないか、あるいは有意に発現量が低い場合に陰性としてもよい。
Muse細胞は、これらの細胞表面の抗原特性に基づいて単離することができる。
When these surface antigens are negative, it means that they are not sorted as positive cells when analyzed using FACS as described above, or that expression is not observed when expression is examined by RT-PCR, Even if it is expressed to such an extent that it cannot be detected by these techniques, it is considered negative in the present invention. In addition, measurement is performed simultaneously with cells such as hematopoietic stem cells that are known to be positive for the above-mentioned marker, and compared to these positive cells, it is hardly detected or may be negative when the expression level is significantly low. .
Muse cells can be isolated based on the antigenic properties of these cell surfaces.

上記のように、Muse細胞は、SSEA-3陽性を指標に単離することができ、さらにCD105の発現を指標に単離することができるが、さらに、NG2、CD34、vWF(フォンビルブランド因子)、c-kit(CD117)、CD146、CD271(NGFR)、Sox10、Snai1、Slug、Tyrp1及びDctからなる群から選択される11個のマーカーのうち少なくとも1個、例えば、2個、3個、4個、5個、6個、7個、8個、9個、10個又は11個のマーカーの非発現を指標に単離することができる。例えば、CD117及びCD146の非発現を指標に単離することができ、さらに、CD117、CD146、NG2、CD34、vWF及びCD271の非発現を指標に単離することができ、さらに、上記の11個のマーカーの非発現を指標に単離することができる。   As described above, Muse cells can be isolated using SSEA-3 positivity as an indicator, and can also be isolated using CD105 expression as an indicator, but also NG2, CD34, vWF (von Willebrand factor) ), C-kit (CD117), CD146, CD271 (NGFR), Sox10, Snai1, Slug, Tyrp1 and Dct, at least one marker, for example, 2, 3, Non-expression of 4, 5, 6, 7, 8, 9, 10, or 11 markers can be isolated using as an indicator. For example, non-expression of CD117 and CD146 can be isolated using as an index, and further, non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be used as an index. The non-expression of the marker can be isolated using as an indicator.

表面マーカーを用いて単離する場合、生体組織から1個又は複数個のMuse細胞、培養等を経ることなく直接単離することが可能である。また、Muse細胞を、細胞形態を顕微鏡等を使って目視することにより同定して単離することが可能である。   In the case of isolation using a surface marker, it is possible to isolate directly from a living tissue without going through one or a plurality of Muse cells, culture or the like. Further, it is possible to identify and isolate Muse cells by visually observing the cell morphology using a microscope or the like.

また、上記のマーカーに加えて、Muse細胞は、他の特定の因子の高発現によっても特徴付けられる。   In addition to the markers described above, Muse cells are also characterized by high expression of other specific factors.

Muse細胞を培養することによりMuse細胞由来の胚様体(EB)様細胞塊が得られる。Muse細胞、無処理細胞、Muse由来胚様体様細胞塊及びヒトES細胞において発現している因子を比較検討することにより、Muse細胞で高発現している因子がわかる。ここで、因子とは遺伝子転写産物、タンパク質、脂質、糖を含む。   By culturing Muse cells, embryonic body (EB) -like cell clusters derived from Muse cells can be obtained. By comparing the factors expressed in Muse cells, untreated cells, Muse-derived embryoid body-like cell clusters, and human ES cells, the factors highly expressed in Muse cells can be determined. Here, factors include gene transcripts, proteins, lipids, and sugars.

Muse細胞においては、以下の18個の因子が高発現している。
(i) SSEA-3
(ii) v-fos FBJ murine osteosarcoma viral oncogene homolog
(iii) solute carrier family 16, member 6 (monocarboxylic acid transporter 7)
(iv) tyrosinase-related protein 1
(v) Calcium channel, voltage-dependent, P/Q type, alpha 1A subunit
(vi) chromosome 16 open reading frame 81
(vii) chitinase 3-like 1 (cartilage glycoprotein-39)
(viii) protease, serine, 35
(ix) kynureninase (L-kynurenine hydrolase)
(x) solute carrier family 16, member 6 (monocarboxylic acid transporter 7)
(xi) apolipoprotein E
(xii) synaptotagmin-like 5
(xiii) chitinase 3-like 1 (cartilage glycoprotein-39)
(xiv) ATP-binding cassette, sub-family A (ABC1), member 13
(xv) angiopoietin-like 4
(xvi) prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)
(xvii) stanniocalcin 1
(xviii) coiled-coil domain containing 102B
In Muse cells, the following 18 factors are highly expressed.
(i) SSEA-3
(ii) v-fos FBJ murine osteosarcoma viral oncogene homolog
(iii) solute carrier family 16, member 6 (monocarboxylic acid transporter 7)
(iv) tyrosinase-related protein 1
(v) Calcium channel, voltage-dependent, P / Q type, alpha 1A subunit
(vi) chromosome 16 open reading frame 81
(vii) chitinase 3-like 1 (cartilage glycoprotein-39)
(viii) protease, serine, 35
(ix) kynureninase (L-kynurenine hydrolase)
(x) solute carrier family 16, member 6 (monocarboxylic acid transporter 7)
(xi) apolipoprotein E
(xii) synaptotagmin-like 5
(xiii) chitinase 3-like 1 (cartilage glycoprotein-39)
(xiv) ATP-binding cassette, sub-family A (ABC1), member 13
(xv) angiopoietin-like 4
(xvi) prostaglandin-endoperoxide synthase 2 (prostaglandin G / H synthase and cyclooxygenase)
(xvii) stanniocalcin 1
(xviii) coiled-coil domain containing 102B

Muse細胞又は多能性幹細胞画分は、上記因子の少なくとも2つ、3つ、4つ、5つ、6つ、7つ、8つ、9つ、10、11、12、13、14、15、16、17又は18が高発現していることを特徴として、少なくとも2つの因子が高発現していることを指標に単離することができる。   Muse cells or pluripotent stem cell fractions are at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15 of the above factors. , 16, 17 or 18 are characterized by high expression, and can be isolated using as an indicator that at least two factors are highly expressed.

また、以下の20個の因子において、ヒトES細胞に対する本発明のMuse細胞の発現量の比が高い。
(a)matrix metallopeptidase 1 (interstitial collagenase)
(b)epiregulin
(c)chitinase 3-like 1 (cartilage glycoprotein-39)
(d)Transcribed locus
(e)chitinase 3-like 1 (cartilage glycoprotein-39)
(f)serglycin
(g)MRNA full length insert cDNA clone EUROIMAGE 1913076
(h) Ras and Rab interactor 2
(i) lumican
(j) CLCA family member 2, chloride channel regulator
(k) interleukin 8
(l) Similar to LOC166075
(m) dermatopontin
(n) EGF, latrophilin and seven transmembrane domain containing 1
(o) insulin-like growth factor binding protein 1
(p) solute carrier family 16, member 4 (monocarboxylic acid transporter 5)
(q) serglycin
(r) gremlin 2, cysteine knot superfamily, homolog (Xenopus laevis)
(s) insulin-like growth factor binding protein 5
(t) sulfide quinone reductase-like (yeast)
Moreover, in the following 20 factors, the ratio of the expression level of the Muse cell of the present invention to the human ES cell is high.
(a) matrix metallopeptidase 1 (interstitial collagenase)
(b) epiregulin
(c) chitinase 3-like 1 (cartilage glycoprotein-39)
(d) Transcribed locus
(e) chitinase 3-like 1 (cartilage glycoprotein-39)
(f) serglycin
(g) MRNA full length insert cDNA clone EUROIMAGE 1913076
(h) Ras and Rab interactor 2
(i) lumican
(j) CLCA family member 2, chloride channel regulator
(k) interleukin 8
(l) Similar to LOC166075
(m) dermatopontin
(n) EGF, latrophilin and seven transmembrane domain containing 1
(o) insulin-like growth factor binding protein 1
(p) solute carrier family 16, member 4 (monocarboxylic acid transporter 5)
(q) serglycin
(r) gremlin 2, cysteine knot superfamily, homolog (Xenopus laevis)
(s) insulin-like growth factor binding protein 5
(t) sulfide quinone reductase-like (yeast)

Muse細胞は、上記因子の少なくとも2つ、3つ、4つ、5つ、6つ、7つ、8つ、9つ、10、11、12、13、14、15、16、17、18、19又は20が高発現していることを特徴として、少なくとも2つの因子が高発現していることを指標に単離することができる。   Muse cells have at least 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, It is characterized by high expression of 19 or 20, and can be isolated using as an indicator that at least two factors are highly expressed.

さらに、Muse細胞は、上記(i)〜(xviii)の因子の少なくとも2つと上記(a)〜(t)の因子の少なくとも2つが同時に高発現していてもよく、これらの遺伝子が高発現していることを指標に単離することができる。   Furthermore, in Muse cells, at least two of the above factors (i) to (xviii) and at least two of the above factors (a) to (t) may be simultaneously highly expressed, and these genes are highly expressed. It can be isolated by using as an index.

さらに、Muse細胞は多能性マーカー以外のオドラント(odorant)受容体(オルファクトリーレセプター; olfactory receptor)群及びケモカイン(chemokine)受容体群の因子を発現していること、すなわち特定のオドラント受容体やケモカイン受容体陽性であることを特徴とする。   Furthermore, Muse cells express factors other than pluripotency markers such as odorant receptors (olfactory receptors) and chemokine receptors, that is, specific odorant receptors and It is characterized by being positive for a chemokine receptor.

Muse細胞で発現しているオドラント受容体として例えば、以下の22個の受容体が挙げられる。
olfactory receptor, family 8, subfamily G, member 2 (OR8G2);
olfactory receptor, family 7, subfamily G, member 3 (OR7G3);
olfactory receptor, family 4, subfamily D, member 5 (OR4D5);
olfactory receptor, family 5, subfamily AP, member 2 (OR5AP2);
olfactory receptor, family 10, subfamily H, member 4 (OR10H4);
olfactory receptor, family 10, subfamily T, member 2 (OR10T2);
olfactory receptor, family 2, subfamily M, member 2 (OR2M2);
olfactory receptor, family 2, subfamily T, member 5 (OR2T5);
olfactory receptor, family 7, subfamily D, member 4 (OR7D4);
olfactory receptor, family 1, subfamily L, member 3 (OR1L3);
olfactory receptor, family 4, subfamily N, member 4 (OR4N4);
olfactory receptor, family 2, subfamily A, member 7 (OR2A7);
guanine nucleotide binding protein (G protein), alpha activating activity polypeptide, olfactory type (GNAL);
olfactory receptor, family 6, subfamily A, member 2 (OR6A2);
olfactory receptor, family 2, subfamily B, member 6 (OR2B6);
olfactory receptor, family 2, subfamily C, member 1 (OR2C1);
olfactory receptor, family 52, subfamily A, member 1 (OR52A1);
olfactory receptor, family 10, subfamily H, member 3 (OR10H3);
olfactory receptor, family 10, subfamily H, member 2 (OR10H2);
olfactory receptor, family 51, subfamily E, member 2 (OR51E2);
olfactory receptor, family 5, subfamily P, member 2 (OR5P2);及び
olfactory receptor, family 10, subfamily P, member 1 (OR10P1)
Examples of odorant receptors expressed in Muse cells include the following 22 receptors.
olfactory receptor, family 8, subfamily G, member 2 (OR8G2);
olfactory receptor, family 7, subfamily G, member 3 (OR7G3);
olfactory receptor, family 4, subfamily D, member 5 (OR4D5);
olfactory receptor, family 5, subfamily AP, member 2 (OR5AP2);
olfactory receptor, family 10, subfamily H, member 4 (OR10H4);
olfactory receptor, family 10, subfamily T, member 2 (OR10T2);
olfactory receptor, family 2, subfamily M, member 2 (OR2M2);
olfactory receptor, family 2, subfamily T, member 5 (OR2T5);
olfactory receptor, family 7, subfamily D, member 4 (OR7D4);
olfactory receptor, family 1, subfamily L, member 3 (OR1L3);
olfactory receptor, family 4, subfamily N, member 4 (OR4N4);
olfactory receptor, family 2, subfamily A, member 7 (OR2A7);
guanine nucleotide binding protein (G protein), alpha activating activity polypeptide, olfactory type (GNAL);
olfactory receptor, family 6, subfamily A, member 2 (OR6A2);
olfactory receptor, family 2, subfamily B, member 6 (OR2B6);
olfactory receptor, family 2, subfamily C, member 1 (OR2C1);
olfactory receptor, family 52, subfamily A, member 1 (OR52A1);
olfactory receptor, family 10, subfamily H, member 3 (OR10H3);
olfactory receptor, family 10, subfamily H, member 2 (OR10H2);
olfactory receptor, family 51, subfamily E, member 2 (OR51E2);
olfactory receptor, family 5, subfamily P, member 2 (OR5P2); and
olfactory receptor, family 10, subfamily P, member 1 (OR10P1)

Muse細胞で発現しているケモカイン受容体としては以下の5個の受容体が挙げられる。
chemokine (C-C motif) receptor 5(CCR5);
chemokine (C-X-C motif) receptor 4(CXCR4);
chemokine (C-C motif) receptor 1(CCR1);
Duffy blood group, chemokine receptor(DARC);及び
chemokine (C-X-C motif) receptor 7(CXCR7)
Examples of chemokine receptors expressed in Muse cells include the following five receptors.
chemokine (CC motif) receptor 5 (CCR5);
chemokine (CXC motif) receptor 4 (CXCR4);
chemokine (CC motif) receptor 1 (CCR1);
Duffy blood group, chemokine receptor (DARC); and
chemokine (CXC motif) receptor 7 (CXCR7)

Muse細胞は、上記の嗅覚受容体の少なくとも1個を発現しており、あるいは、上記のケモカイン受容体の少なくとも1個を発現している。   Muse cells express at least one of the above olfactory receptors or express at least one of the above chemokine receptors.

これらのオドラント受容体やケモカイン受容体と受容体に結合する遊走因子の作用でMuse細胞は、損傷組織へ遊走し、生着し、その場で分化する。例えば、肝臓、皮膚、脊髄、筋肉が損傷した場合、特定の遊走因子と細胞表面に発現しているオドラント受容体の働きで、それぞれの組織に遊走し、生着し、肝臓(内胚葉)、皮膚(外胚葉)、脊髄(外胚葉)、筋肉(中胚葉)細胞に分化し、組織を再生することができる。   Muse cells migrate to the damaged tissue, engraft, and differentiate on the spot by the action of these odorant receptors and chemokine receptors and migratory factors that bind to the receptors. For example, when the liver, skin, spinal cord, or muscle is damaged, the specific migratory factor and the odorant receptor expressed on the cell surface migrate to and engraft each tissue, the liver (endoderm), Differentiated into skin (ectodermal), spinal cord (ectodermal), muscle (mesoderm) cells, and tissue can be regenerated.

さらに、Muse細胞において、Rex1、Sox2、KLF-4、c-Myc、DPPA2、ERAS、GRB7、SPAG9、TDGF1等がアップレギュレートされており、Muse細胞の細胞塊において、DAZL、DDX4、DPPA4、Stella、Hoxb1、PRDM1、SPRY2等がアップレギュレートされている。   Furthermore, Rex1, Sox2, KLF-4, c-Myc, DPPA2, ERAS, GRB7, SPAG9, TDGF1, etc. are up-regulated in Muse cells, and DAZL, DDX4, DPPA4, Stella, etc. , Hoxb1, PRDM1, SPRY2, etc. are up-regulated.

また、Muse細胞においては、造血幹細胞マーカーであるCD34及びCD117の発現は認めらないかもしくは発現が極めて低い。   Further, in Muse cells, expression of CD34 and CD117, which are hematopoietic stem cell markers, is not observed or is very low.

さらに、Muse細胞は、生体の中胚葉系組織又は間葉系組織等の細胞に細胞ストレスをかけ、生き残った細胞を回収することにより単離することができる。ここで、細胞ストレスとは外的ストレスをいい、プロテアーゼ処理、低酸素条件下での培養、低リン酸条件下での培養、血清飢餓状態での培養、糖飢餓状態での培養、放射線曝露下での培養、熱ショックへの曝露下での培養、有害物質存在下での培養、活性酸素存在下での培養、機械的刺激下での培養、圧力処理下での培養等によりストレスに曝露することをいう。この中でもプロテアーゼ処理、すなわちプロテアーゼ存在下での培養が好ましい。プロテアーゼは限定されず、トリプシン、キモトリプシン等のセリンプロテアーゼ、ペプシン等のアスパラギン酸プロテアーゼ、パパイン、キモパパイン等のシステインプロテアーゼ、サーモリシン等の金属プロテアーゼ、グルタミン酸プロテアーゼ、N-末端スレオニンプロテアーゼなどを用いることができる。プロテアーゼを培養に添加する際の添加濃度は限定されず、一般的にシャーレ等で培養した付着細胞を剥がすときに用いる濃度で用いればよい。Muse細胞は、上記外的ストレスに耐性を有する幹細胞、例えば、トリプシンに耐性を有する細胞ということができる。   Furthermore, Muse cells can be isolated by applying cell stress to cells such as mesodermal or mesenchymal tissues in a living body and collecting surviving cells. Here, cell stress refers to external stress, including protease treatment, culture under low oxygen conditions, culture under low phosphate conditions, culture under serum starvation conditions, culture under sugar starvation conditions, under radiation exposure Exposure to stress by culturing in the presence of heat shock, culturing in the presence of toxic substances, culturing in the presence of harmful substances, culturing in the presence of active oxygen, culturing under mechanical stimulation, culturing under pressure treatment, etc. That means. Among these, protease treatment, that is, culture in the presence of protease is preferable. The protease is not limited, and serine proteases such as trypsin and chymotrypsin, aspartic proteases such as pepsin, cysteine proteases such as papain and chymopapain, metalloproteases such as thermolysin, glutamate protease, N-terminal threonine protease and the like can be used. The concentration at which protease is added to the culture is not limited, and it may be used at a concentration generally used when peeling adherent cells cultured in a petri dish or the like. Muse cells can be referred to as stem cells that are resistant to the above external stress, for example, cells that are resistant to trypsin.

生体の中胚葉系組織又は間葉系組織等は限定されず、骨髄単核細胞、皮膚細胞等の線維芽細胞画分、歯髄組織、眼球組織、毛根組織等が含まれる。細胞としては、培養細胞も組織から採取した細胞も用いることもできる。この中でも、骨髄細胞、皮膚細胞が望ましく、例えば、ヒト骨髄間葉系細胞(MSC)画分又はヒト皮膚線維芽細胞画分が挙げられる。骨髄間葉系細胞画分は、骨髄穿刺液を2〜3週間培養することにより得ることができる。   Biological mesodermal or mesenchymal tissues are not limited, and include bone marrow mononuclear cells, fibroblast fractions such as skin cells, dental pulp tissues, eyeball tissues, hair root tissues, and the like. As the cells, cultured cells or cells collected from tissues can be used. Among these, bone marrow cells and skin cells are desirable, and examples thereof include a human bone marrow mesenchymal cell (MSC) fraction and a human skin fibroblast fraction. The bone marrow mesenchymal cell fraction can be obtained by culturing a bone marrow puncture solution for 2 to 3 weeks.

上記の各種のストレスを受けた組織の細胞の大部分は死滅し、生き残った細胞中にMuse細胞が含まれる。細胞にストレスをかけたのち、死細胞を除去する必要があるが、プロテアーゼを用いた場合は、これらの死細胞はプロテアーゼの作用により分解される。   Most of the cells of the above-mentioned various stressed tissues are killed, and the surviving cells include Muse cells. It is necessary to remove dead cells after applying stress to the cells. When protease is used, these dead cells are degraded by the action of the protease.

また、細胞にストレスをかけた後に、細胞に物理的衝撃を与え壊れ易くなった細胞を除去してもよい。物理的衝撃は、例えば激しいピペッティング、激しい攪拌、ボルテックス等により与えることができる。   Further, after applying stress to the cells, the cells that have been subjected to a physical impact on the cells and have become fragile may be removed. The physical impact can be applied, for example, by vigorous pipetting, vigorous stirring, vortexing or the like.

細胞に細胞ストレスをかけ、必要に応じて物理的衝撃を与えた後に、細胞群を遠心分離にかけ、生き残った細胞をペレットとして得て回収することにより、Muse細胞を単離することができる。また、このようにして得られた細胞からさらに、下記の表面マーカーを指標にMuse細胞又は多能性細胞画分を単離することもできる。   Muse cells can be isolated by applying cell stress to the cells and applying physical impact as necessary, and then centrifuging the cells to obtain and collect the surviving cells as pellets. In addition, Muse cells or pluripotent cell fractions can also be isolated from the cells thus obtained using the following surface markers as indicators.

また、外傷や火傷等のストレスを受けた生体の中胚葉系組織又は間葉系組織等を培養し、遊走した細胞を回収してもMuse細胞を単離することができる。傷害を受けた組織の細胞はストレスに曝露されるので、本発明においては、傷害を受けた生体の中胚葉系組織又は間葉系組織等を培養することも生体の中胚葉系組織又は間葉系組織細胞等に細胞ストレスをかけるという。   Also, Muse cells can be isolated by culturing mesodermal or mesenchymal tissues or the like that have been subjected to stress such as trauma or burn, and collecting the migrated cells. Since cells of the injured tissue are exposed to stress, in the present invention, injured mesodermal tissue or mesenchymal tissue or the like may be cultured in the living organism. It is said that cell stress is applied to systemic tissue cells.

一例として、これらの細胞をトリプシン処理する方法について説明する。このときのトリプシン濃度は、限定されないが、例えば接着細胞の通常の培養において、培養容器に接着した接着培養を剥がすときに用いられる濃度範囲で用いればよく、0.1〜1%、好ましくは0.1〜0.5%が例示される。例えば、10〜50万個の細胞を含む生体の中胚葉系組織又は間葉系組織等由来の細胞を上記濃度のトリプシン溶液5ml中でインキュベーションすることにより外的ストレスに曝すことができる。トリプシン処理時間は、5〜24時間、好ましくは5〜20時間程度である。本発明においては、8時間以上のトリプシン処理、例えば8時間又は16時間の処理を長時間トリプシン処理という。   As an example, a method for treating these cells with trypsin will be described. The trypsin concentration at this time is not limited. For example, in the normal culture of adherent cells, the trypsin concentration may be used within the concentration range used when peeling the adherent culture adhered to the culture vessel, and is 0.1 to 1%, preferably 0.1 to 0.5. % Is exemplified. For example, cells derived from mesodermal or mesenchymal tissue containing 100 to 500,000 cells can be exposed to external stress by incubating in 5 ml of a trypsin solution having the above concentration. The trypsin treatment time is 5 to 24 hours, preferably about 5 to 20 hours. In the present invention, trypsin treatment for 8 hours or more, for example, treatment for 8 hours or 16 hours is referred to as long-time trypsin treatment.

トリプシン処理後、上記のように、ピペッティング、攪拌、ボルテックス等により物理的衝撃を与えることが望ましい。それは死んだ細胞あるいは死にかけている細胞を除去するためである。   After the trypsin treatment, it is desirable to give a physical impact by pipetting, stirring, vortexing or the like as described above. This is to remove dead or dying cells.

トリプシン処理後の浮遊培養の際には細胞同士の凝集を防ぐために、例えば、メチルセルロースゲル等のゲル中でインキュベーションするのが望ましい。また、細胞の培養容器への付着を防ぎ浮遊状態を維持するために、容器をPoly(2-hydroxyethyl methacrylate)等でコートしておくことが望ましい。   In suspension culture after trypsin treatment, in order to prevent aggregation between cells, it is desirable to incubate in a gel such as methylcellulose gel. In addition, it is desirable to coat the container with Poly (2-hydroxyethyl methacrylate) or the like in order to prevent the cells from adhering to the culture container and maintain the floating state.

外的ストレスに曝した細胞を遠心分離により集め培養を行うと細胞塊(細胞クラスター)を形成する。この細胞塊の大きさは直径25μmから150μm程度である。Muse細胞は、この外的ストレスに曝して生き残った細胞集団中に濃縮した状態で含まれる。この細胞集団を富Muse細胞画分(Muse enriched population)と呼ぶ。富Muse細胞画分中のMuse細胞の存在割合は、ストレス処理の方法により異なる。   When cells exposed to external stress are collected by centrifugation and cultured, cell clusters (cell clusters) are formed. The size of this cell mass is about 25 μm to 150 μm in diameter. Muse cells are contained in a concentrated state in the cell population that survived this external stress. This cell population is called the Muse enriched population. The proportion of Muse cells present in the rich Muse cell fraction varies depending on the stress treatment method.

このようにMuse細胞がストレスをかけた後も生存することは、Muse細胞がストレス耐性であることを示している。   Thus, the survival of the Muse cells after applying stress indicates that the Muse cells are resistant to stress.

生体の中胚葉系組織又は間葉系組織等由来の細胞の培養に用いる培地、培養条件は通常の動物細胞の培養で用いる培地、培養条件を採用すればよい。また、公知の幹細胞培養用培地を用いてもよい。培地には、適宜ウシ胎児血清等の血清やペニシリン、ストレプトマイシン等の抗生物質及び種々の生理活性物質を添加してもよい。   The medium and culture conditions used for culturing cells derived from mesodermal or mesenchymal tissues of a living body may be those employed for normal animal cell culture. Further, a known stem cell culture medium may be used. To the medium, serum such as fetal bovine serum, antibiotics such as penicillin and streptomycin, and various physiologically active substances may be appropriately added.

さらに、Muse細胞は、Muse細胞の派生細胞又は誘導細胞である多能性幹細胞も含む。派生細胞又は誘導細胞とはMuse細胞を培養して得られる細胞又は細胞群、あるいはMuse細胞に外来遺伝子の導入等の人為的な誘導操作を行い得られる細胞をいい、子孫細胞も含む。なお、本発明時点において報告されているiPS細胞は、皮膚線維芽細胞などの生体組織の分化した細胞に外来遺伝子導入等することによりリプログラミングした結果、多能性幹細胞に誘導された細胞といわれており、本発明の組織から直接得ることができ、すでに多能性幹細胞としての性質を有する細胞に外来遺伝子導入等の人為的な誘導操作を行い得られた細胞は、iPS細胞と区別される。   Furthermore, Muse cells also include pluripotent stem cells that are derived or derived from Muse cells. Derived cells or induced cells refer to cells or cell groups obtained by culturing Muse cells, or cells obtained by performing artificial induction operations such as introduction of foreign genes into Muse cells, including progeny cells. The iPS cells reported at the time of the present invention are said to be cells induced by pluripotent stem cells as a result of reprogramming by introducing a foreign gene into differentiated cells of living tissue such as skin fibroblasts. Cells that can be obtained directly from the tissue of the present invention and have already been subjected to artificial induction procedures such as introduction of foreign genes into cells that already have the properties of pluripotent stem cells are distinguished from iPS cells. .

Muse細胞は、Muse細胞を浮遊培養することにより得られる胚様体様(Embryoid body(EB body)-like)細胞塊も含む。胚様体は、Muse細胞を浮遊培養することにより、細胞塊として形成される。この際、Muse細胞を培養することにより得られる胚様体をMuse細胞由来胚様体様細胞塊と呼ぶことがある(Mクラスター(M-cluster)と呼ぶこともある)。胚様体様細胞塊を形成するための浮遊培養の方法として、メチルセルロース等の水溶性ポリマーを含有した培地を用いた培養(Nakahata, T. et al., Blood 60, 352-361 (1982))やハンギングドロップ培養(Keller,J.Physiol.(Lond)168:131-139,1998)等が挙げられる。さらに、Muse細胞は、前記胚様体様細胞塊からセルフリニューアルして得られる胚様体様細胞塊及び胚様体様細胞塊に含まれる細胞及び多能性幹細胞も包含する。ここで、セルフリニューアルとは、胚様体様細胞塊に含まれる細胞を培養し、再度胚様体様細胞塊を形成させることをいう。セルフリニューアルは1〜複数回のサイクルを繰り返せばよい。また、Muse細胞は前記いずれかの胚様体様細胞塊及び胚様体様細胞塊に含まれる細胞から分化した細胞及び組織も包含する。   Muse cells also contain embryoid body-like (Embryoid body (EB body) -like) cell mass obtained by suspension culture of Muse cells. The embryoid body is formed as a cell mass by suspension culture of Muse cells. In this case, an embryoid body obtained by culturing Muse cells is sometimes referred to as a Muse cell-derived embryoid body-like cell cluster (sometimes referred to as an M-cluster). Culture using a medium containing a water-soluble polymer such as methylcellulose (Nakahata, T. et al., Blood 60, 352-361 (1982)) And hanging drop culture (Keller, J. Physiol. (Lond) 168: 131-139, 1998). Further, the Muse cell includes an embryoid body-like cell cluster obtained by self-renewal from the embryoid body-like cell cluster, cells contained in the embryoid body-like cell cluster, and pluripotent stem cells. Here, self-renewal refers to culturing cells contained in an embryoid body-like cell cluster to form an embryoid body-like cell cluster again. The self-renewal may be repeated one to several times. The Muse cells also include cells and tissues differentiated from any of the aforementioned embryoid body-like cell clusters and cells contained in the embryoid body-like cell clusters.

他家移植とは、他人(別個体)の組織や細胞を移植することをいう。
Muse細胞は、表面にHLA(Human Lymphocyte antigen)のうち、HLA class I抗原は発現しているが、HLA classII抗原を発現していない。HLA classII抗原を有する組織や細胞を他家移植した場合、ドナー組織又は細胞のHLA class II抗原による抗原提示により細胞性免疫が活性化され拒絶される。従って、HLA class II抗原を発現していないMuse細胞を他家に移植した場合であっても、拒絶反応が起こりにくく、生着し得る。このため、移植時に免疫抑制剤を用いる必要がない。
Transplantation refers to transplanting tissue or cells of another person (separate body).
Muse cells express HLA class I antigen among HLA (Human Lymphocyte antigen) on the surface, but do not express HLA class II antigen. When a tissue or cell having an HLA class II antigen is transplanted to another family, cellular immunity is activated and rejected by antigen presentation by the donor tissue or cell with the HLA class II antigen. Therefore, even when Muse cells that do not express the HLA class II antigen are transplanted into another family, rejection is unlikely to occur and they can be engrafted. For this reason, it is not necessary to use an immunosuppressive agent at the time of transplantation.

Muse細胞は、pluripotencyを有しており、あらゆる組織へと分化し得る。従って、Muse細胞は、再生医療等に用いることができ、機能が損なわれた組織等にMuse細胞を補う細胞治療を施すことにより組織が再生し得る。例えば、各種組織、各種器官等の再生に用いることができる。具体的には皮膚、脳脊髄、肝臓、筋肉等が挙げられる。Muse細胞を損傷あるいは障害を受けた組織、器官等に直接あるいは近傍に投与することにより、Muse細胞はその組織、器官内に侵入し、その組織特有の細胞に分化し、組織、器官の再生、再建に貢献し得る。また、静脈投与等により全身投与してもよい。この場合、Muse細胞は、例えば、損傷を受けた組織や器官をホーミング等により指向し、そこに到達・侵入した上で、その組織や器官の細胞に分化し、組織、器官の再生、再建に貢献し得る。
すなわち、Muse細胞は再生医療のための他家移植用の細胞治療に用いることができる。
Muse cells have pluripotency and can differentiate into any tissue. Therefore, Muse cells can be used for regenerative medicine and the like, and the tissue can be regenerated by applying cell therapy to supplement the Muse cells to a tissue with impaired function. For example, it can be used for regeneration of various tissues and various organs. Specific examples include skin, cerebral spinal cord, liver, and muscle. By administering Muse cells directly or nearby to damaged or damaged tissues, organs, etc., Muse cells invade the tissues, organs, differentiate into cells specific to the tissues, regeneration of tissues, organs, Can contribute to reconstruction. Further, systemic administration may be performed by intravenous administration or the like. In this case, for example, the Muse cell is directed to a damaged tissue or organ by homing, etc., and reaches / invades, and then differentiates into a cell of the tissue or organ to regenerate or reconstruct the tissue or organ. Can contribute.
That is, Muse cells can be used for cell therapy for allogeneic transplantation for regenerative medicine.

投与は、例えば皮下注、静注、筋注、腹腔内注等の非経口投与や経口投与、あるいは胚への子宮内注射等により行うことができる。また、局所投与でも全身投与でもよい。局所投与は例えばカテーテルを利用して行うことができる。投与量は、再生しようとする器官、組織の種類や、サイズにより適宜決定することができる。   Administration can be performed, for example, by parenteral or oral administration such as subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, or intrauterine injection into an embryo. Moreover, local administration or systemic administration may be sufficient. Local administration can be performed using a catheter, for example. The dose can be appropriately determined depending on the type and size of the organ or tissue to be regenerated.

再生しようとする器官は限定されず、骨髄、脊髄、血液、脾臓、肝臓、肺、腸管、眼、脳、免疫系、循環系、骨、結合組織、筋、心臓、血管、膵臓、中枢神経系、末梢神経系、腎臓、膀胱、皮膚、上皮付属器、乳房−乳腺、脂肪組織、角膜、および口、食道、膣、肛門を含む粘膜等を含む。また、治療対象となる疾患として、癌、心血管疾患、代謝疾患、肝疾患、糖尿病、肝炎、血友病、血液系疾患、脊髄損傷等の変性または外傷性神経疾患、自己免疫疾患、遺伝的欠陥、結合組織疾患、貧血、感染症、移植拒絶、虚血、炎症、皮膚や筋肉の損傷等が挙げられる。   The organ to be regenerated is not limited, but bone marrow, spinal cord, blood, spleen, liver, lung, intestinal tract, eye, brain, immune system, circulatory system, bone, connective tissue, muscle, heart, blood vessel, pancreas, central nervous system , Peripheral nervous system, kidney, bladder, skin, epithelial appendages, breast-mammary gland, adipose tissue, cornea, and mucous membranes including mouth, esophagus, vagina, anus, and the like. Diseases to be treated include cancer, cardiovascular disease, metabolic disease, liver disease, diabetes, hepatitis, hemophilia, blood system disease, degenerative or traumatic neurological diseases such as spinal cord injury, autoimmune disease, genetic Examples include defects, connective tissue diseases, anemia, infections, transplant rejection, ischemia, inflammation, and skin and muscle damage.

細胞は医薬として許容される基材と共に投与してもよい。該基材は例えばコラーゲン等でできた生体親和性が高い物質や、生分解性の物質できており、粒子状、板状、筒状、容器状等の形状とすればよく、細胞を該基材に結合させあるいは該基材中に収容して投与すればよい。   The cells may be administered with a pharmaceutically acceptable substrate. The substrate is made of, for example, collagen, a highly biocompatible substance, or a biodegradable substance, and may be in the form of particles, plates, cylinders, containers, and the like. What is necessary is just to couple | bond with a material or to accommodate in this base material and to administer.

また、Muse細胞をin vitroで分化誘導し、さらに分化した細胞を用いて組織を構築させ、該分化した細胞又は該組織を他家移植してもよい。Muse細胞は、腫瘍化しないので、移植した前記分化した細胞又は該組織にMuse細胞が未分化のまま含まれていても癌化の可能性が低く安全である。   Alternatively, differentiation of Muse cells may be induced in vitro, a tissue may be constructed using the differentiated cells, and the differentiated cells or the tissue may be transplanted to another family. Since Muse cells do not become tumorous, even if the transplanted differentiated cells or the tissues contain undifferentiated Muse cells, the possibility of canceration is low and safe.

さらに、Muse細胞を組織の変性や機能不全を原因とする疾患の治療に用いることができる。この場合、例えば、Muse細胞をex vivoで濃縮し、増殖させ、あるいは分化させて体内に戻せばよく、例えば、Muse細胞を特定の組織の細胞に分化させ、該細胞を治療しようとする組織に移植すればよい。また、細胞の移植により、in situ 細胞治療を行うこともできる。この場合、対象細胞の例として、肝臓細胞、神経細胞やグリア細胞などの神経系細胞、皮膚細胞、骨格筋細胞などの筋肉細胞が挙げられ、Muse細胞をこれらの細胞に分化させ、移植し、in situで治療を行うことができる。該治療により、例えば、パーキンソン病、脳梗塞、脊髄損傷、筋変性疾患などを治療することができる。Muse細胞は、腫瘍化しないので、このような治療に用いても癌化の可能性が低く安全である。   Furthermore, Muse cells can be used for the treatment of diseases caused by tissue degeneration or dysfunction. In this case, for example, the Muse cells may be concentrated ex vivo, proliferated, or differentiated and returned to the body. For example, the Muse cells are differentiated into cells of a specific tissue and the cells are treated into the tissue to be treated. Just transplant. In situ cell therapy can also be performed by cell transplantation. In this case, examples of target cells include liver cells, nervous cells such as nerve cells and glial cells, skin cells, muscle cells such as skeletal muscle cells, Muse cells are differentiated into these cells, transplanted, Treatment can be performed in situ. By this treatment, for example, Parkinson's disease, cerebral infarction, spinal cord injury, muscle degenerative disease and the like can be treated. Muse cells do not become tumors, so even if they are used for such treatment, they are safe with no possibility of canceration.

また、Muse細胞を、分化させて血液や血液成分を形成させることにより、血液や血液成分をex vivo、in vitroで形成させることができる、血液成分として、赤血球、白血球、血小板等が挙げられる。このようにして形成させた血液や血液成分を、他家輸血に用いることができる。   In addition, blood and blood components can be formed ex vivo and in vitro by differentiating Muse cells to form blood and blood components. Examples of blood components include red blood cells, white blood cells, and platelets. The blood and blood components thus formed can be used for cross-family transfusion.

上記のように、Muse細胞を治療に用いる場合、ex vivo、in vivo、in vitroのいずれで分化させてもよい。Muse細胞は、例えば、骨芽細胞、軟骨細胞、脂肪細胞、線維芽細胞、骨髄間質、骨格筋、平滑筋、心筋、眼、内皮、上皮、肝、膵、造血、グリア、神経細胞、稀突起膠細胞等に分化する。Muse細胞の分化は、分化因子の存在下で、培養することにより達成することができる。分化因子としては、塩基性繊維芽細胞成長因子(bFGF)、血管内皮成長因子(VEGF)、ジメチルスルホキシド(DMSO)およびイソプロテレノール;あるいは繊維芽細胞成長因子4(FGF4)、肝細胞成長因子(HGF)等が挙げられる。   As described above, when Muse cells are used for treatment, they may be differentiated ex vivo, in vivo, or in vitro. Muse cells are, for example, osteoblasts, chondrocytes, adipocytes, fibroblasts, bone marrow stroma, skeletal muscle, smooth muscle, myocardium, eyes, endothelium, epithelium, liver, pancreas, hematopoiesis, glia, nerve cells, rare Differentiate into oligodendrocytes. Differentiation of Muse cells can be achieved by culturing in the presence of differentiation factors. Differentiation factors include basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), dimethyl sulfoxide (DMSO) and isoproterenol; or fibroblast growth factor 4 (FGF4), hepatocyte growth factor ( HGF).

Muse細胞を治療に用いる場合、タンパク質性の抗癌物質や生理活性物質等をコードする遺伝子を導入してもよい。これにより、Muse細胞は、治療薬のデリバリー機能も有することになる。このような物質として例えば、抗血管新生薬が挙げられる。   When Muse cells are used for treatment, genes encoding proteinaceous anticancer substances, physiologically active substances and the like may be introduced. As a result, the Muse cells also have a therapeutic drug delivery function. An example of such a substance is an anti-angiogenic drug.

本発明は、Muse細胞、Muse細胞からできた胚様体様細胞塊、及びMuse細胞や前記胚様体様細胞塊から分化させて得られた細胞若しくは組織・器官を含む、他家移植用細胞治療用組成物若しくは他家移植用細胞治療用組成物を包含する。これらを他家移植用再生医療用材料若しくは他家移植用再生医療用組成物ということもできる。該組成物はMuse細胞、Muse細胞からできた胚様体様細胞塊、又はMuse細胞や前記胚様体様細胞塊から分化させて得られた細胞若しくは組織・器官に加えて、医薬的に許容される緩衝液や希釈液等を含む。   The present invention relates to Muse cells, embryoid body-like cell clusters made from Muse cells, and cells for transplantation to other plants, including Muse cells and cells or tissues / organs obtained by differentiation from the embryoid body-like cell clusters. It includes a therapeutic composition or a cell therapeutic composition for allogeneic transplantation. These can also be referred to as regenerative medical materials for allogeneic transplantation or regenerative medical compositions for allogeneic transplantation. The composition is pharmaceutically acceptable in addition to Muse cells, embryoid body-like cell clusters made from Muse cells, or cells or tissues / organs obtained by differentiation from Muse cells or the embryoid body-like cell clusters. Including buffer solution and diluent.

本発明を以下の実施例によって具体的に説明するが、本発明はこれらの実施例によって限定されるものではない。   The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

実施例1
ヒト骨髄間葉系細胞由来のSSEA-3陽性細胞のHLA class I及びHLA class II抗原の発現をフローサイトメトリーにより確認した。図1に結果を示す。図1に示すように、ヒト骨髄間葉系細胞においては、HLA1は陽性であったが、HLA2は陰性であった。
Example 1
Expression of HLA class I and HLA class II antigens in SSEA-3 positive cells derived from human bone marrow mesenchymal cells was confirmed by flow cytometry. The results are shown in FIG. As shown in FIG. 1, in human bone marrow mesenchymal cells, HLA1 was positive, but HLA2 was negative.

同様にヒト線維芽細胞由来のSSEA-3陽性細胞のHLA class I及びHLA class II抗原の発現を確信した。図2に結果を示す。図2に示すように、ヒト線維芽細胞においても、HLA1は陽性であったが、HLA2は陰性であった。   Similarly, expression of HLA class I and HLA class II antigens of SSEA-3 positive cells derived from human fibroblasts was confirmed. The results are shown in FIG. As shown in FIG. 2, in human fibroblasts, HLA1 was positive but HLA2 was negative.

実施例2
抗ヒトHLA-ABC抗体(eBioscience)を用いた免疫細胞化学染色により(2次抗体としては、抗マウスIgG抗体(Alexa568標識を使用)、Muse細胞(SSEA-3陽性)及び非Muse細胞(SSEA-3陰性)におけるHLA Class Iの発現を調べた。結果を図3に示す。図3に示すように、Muse細胞及び非Muse細胞のいずれにおいても、MHC class Iの発現が認められた。
Example 2
By immunocytochemical staining using anti-human HLA-ABC antibody (eBioscience) (as secondary antibody, anti-mouse IgG antibody (using Alexa568 label), Muse cells (SSEA-3 positive) and non-Muse cells (SSEA- 3), the expression of HLA Class I was examined, and the results are shown in Fig. 3. As shown in Fig. 3, expression of MHC class I was observed in both Muse cells and non-Muse cells.

抗ヒトHLA-DR抗体(eBioscience)を用いた免疫細胞化学染色により(2次抗体としては、抗マウスIgG抗体(Alexa568標識を使用)、Muse細胞(SSEA-3陽性)及び非Muse細胞(SSEA-3陰性)におけるHLA Class IIの発現を調べた。結果を図4に示す。図4に示すように、Muse細胞及び非Muse細胞のいずれにおいても、MHC class IIの発現は認められなかった。   By immunocytochemical staining using anti-human HLA-DR antibody (eBioscience) (as secondary antibody, anti-mouse IgG antibody (using Alexa568 label), Muse cells (SSEA-3 positive) and non-Muse cells (SSEA- 3), the expression of HLA Class II was examined, and the results are shown in Fig. 4. As shown in Fig. 4, expression of MHC class II was not observed in both Muse cells and non-Muse cells.

図5は、上記実験における非特異的反応を示す図である。図5に示すように、Alexa568による発光は認められなかった。これは、非特異的反応がないことを示す。   FIG. 5 is a diagram showing a nonspecific reaction in the experiment. As shown in FIG. 5, light emission by Alexa568 was not recognized. This indicates that there is no non-specific reaction.

Claims (14)

生体組織から単離できるSSEA-3陽性であって、HLA class II抗原を発現しない多能性幹細胞を含む、他家移植用細胞治療用組成物。   A composition for cell therapy for allogeneic transplantation, comprising pluripotent stem cells that are SSEA-3 positive and that do not express an HLA class II antigen that can be isolated from a living tissue. さらに、多能性幹細胞がCD105陽性である、請求項1記載の他家移植用細胞治療用組成物。   Furthermore, the composition for cell therapy for allogeneic transplantation of Claim 1 whose pluripotent stem cell is CD105 positive. さらに、多能性幹細胞がCD117陰性及びCD146陰性である、請求項1又は2に記載の他家移植用細胞治療用組成物。   Furthermore, the composition for cell transplantation for allogeneic transplantation according to claim 1 or 2, wherein the pluripotent stem cells are CD117 negative and CD146 negative. さらに、多能性幹細胞がCD117陰性、CD146陰性、NG2陰性、CD34陰性、vWF陰性及びCD271陰性である、請求項1又は2に記載の他家移植用細胞治療用組成物。   Furthermore, the composition for cell transplantation for allogeneic transplantation according to claim 1 or 2, wherein the pluripotent stem cells are CD117 negative, CD146 negative, NG2 negative, CD34 negative, vWF negative and CD271 negative. さらに、多能性幹細胞がCD34陰性、CD117陰性、CD146陰性、CD271陰性、NG2陰性、vWF陰性、Sox10陰性、Snail陰性、Slug陰性、Tyrp1陰性及びDct陰性である、請求項1又は2に記載の他家移植用細胞治療用組成物。   Furthermore, pluripotent stem cells are CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snail negative, Slug negative, Tyrp1 negative and Dct negative according to claim 1 or 2. A cell therapy composition for allogeneic transplantation. さらに、多能性幹細胞がテロメラーゼ活性が低いか又は無い、請求項1〜5のいずれか1項に記載の他家移植用細胞治療用組成物。   Furthermore, the composition for cell therapy for allogeneic transplantation according to any one of claims 1 to 5, wherein the pluripotent stem cells have low or no telomerase activity. さらに、多能性幹細胞が三胚葉に分化する能力を持つ、請求項1〜6のいずれか1項に記載の他家移植用細胞治療用組成物。   Furthermore, the composition for cell therapy for allogeneic transplantation of any one of Claims 1-6 which has the capability in which a pluripotent stem cell differentiates into three germ layers. さらに、多能性幹細胞が腫瘍性増殖を示さない、請求項1〜7のいずれか1項に記載の他家移植用細胞治療用組成物。   Furthermore, the composition for cell therapy for allogeneic transplantation of any one of Claims 1-7 in which a pluripotent stem cell does not show neoplastic proliferation. さらに、多能性幹細胞がセルフリニューアル能を持つ、請求項1〜8のいずれか1項に記載の他家移植用細胞治療用組成物。   Furthermore, the composition for cell therapy for allogeneic transplantation of any one of Claims 1-8 in which a pluripotent stem cell has self-renewal ability. さらに、多能性幹細胞がストレス耐性である、請求項1〜9のいずれか1項に記載の他家移植用細胞治療用組成物。   Furthermore, the composition for cell transplantation for xenotransplantation of any one of Claims 1-9 whose pluripotent stem cell is stress tolerance. さらに、多能性幹細胞が貪食能が高い、請求項1〜10のいずれか1項に記載の他家移植用細胞治療用組成物。   Furthermore, the composition for cell transplantation for autologous transplantation of any one of Claims 1-10 in which a pluripotent stem cell has high phagocytic ability. さらに、多能性幹細胞が以下に示す22個のオドラント受容体の少なくとも一つが陽性の請求項1〜11のいずれか1項に記載の他家移植用細胞治療用組成物:
olfactory receptor, family 8, subfamily G, member 2 (OR8G2);
olfactory receptor, family 7, subfamily G, member 3 (OR7G3);
olfactory receptor, family 4, subfamily D, member 5 (OR4D5);
olfactory receptor, family 5, subfamily AP, member 2 (OR5AP2);
olfactory receptor, family 10, subfamily H, member 4 (OR10H4);
olfactory receptor, family 10, subfamily T, member 2 (OR10T2);
olfactory receptor, family 2, subfamily M, member 2 (OR2M2);
olfactory receptor, family 2, subfamily T, member 5 (OR2T5);
olfactory receptor, family 7, subfamily D, member 4 (OR7D4);
olfactory receptor, family 1, subfamily L, member 3 (OR1L3);
olfactory receptor, family 4, subfamily N, member 4 (OR4N4);
olfactory receptor, family 2, subfamily A, member 7 (OR2A7);
guanine nucleotide binding protein (G protein), alpha activating activity polypeptide, olfactory type (GNAL);
olfactory receptor, family 6, subfamily A, member 2 (OR6A2);
olfactory receptor, family 2, subfamily B, member 6 (OR2B6);
olfactory receptor, family 2, subfamily C, member 1 (OR2C1);
olfactory receptor, family 52, subfamily A, member 1 (OR52A1);
olfactory receptor, family 10, subfamily H, member 3 (OR10H3);
olfactory receptor, family 10, subfamily H, member 2 (OR10H2);
olfactory receptor, family 51, subfamily E, member 2 (OR51E2);
olfactory receptor, family 5, subfamily P, member 2 (OR5P2);及び
olfactory receptor, family 10, subfamily P, member 1 (OR10P1)。
Furthermore, the composition for cell transplantation for transplantation according to any one of claims 1 to 11, wherein at least one of the 22 odorant receptors shown below is positive for pluripotent stem cells:
olfactory receptor, family 8, subfamily G, member 2 (OR8G2);
olfactory receptor, family 7, subfamily G, member 3 (OR7G3);
olfactory receptor, family 4, subfamily D, member 5 (OR4D5);
olfactory receptor, family 5, subfamily AP, member 2 (OR5AP2);
olfactory receptor, family 10, subfamily H, member 4 (OR10H4);
olfactory receptor, family 10, subfamily T, member 2 (OR10T2);
olfactory receptor, family 2, subfamily M, member 2 (OR2M2);
olfactory receptor, family 2, subfamily T, member 5 (OR2T5);
olfactory receptor, family 7, subfamily D, member 4 (OR7D4);
olfactory receptor, family 1, subfamily L, member 3 (OR1L3);
olfactory receptor, family 4, subfamily N, member 4 (OR4N4);
olfactory receptor, family 2, subfamily A, member 7 (OR2A7);
guanine nucleotide binding protein (G protein), alpha activating activity polypeptide, olfactory type (GNAL);
olfactory receptor, family 6, subfamily A, member 2 (OR6A2);
olfactory receptor, family 2, subfamily B, member 6 (OR2B6);
olfactory receptor, family 2, subfamily C, member 1 (OR2C1);
olfactory receptor, family 52, subfamily A, member 1 (OR52A1);
olfactory receptor, family 10, subfamily H, member 3 (OR10H3);
olfactory receptor, family 10, subfamily H, member 2 (OR10H2);
olfactory receptor, family 51, subfamily E, member 2 (OR51E2);
olfactory receptor, family 5, subfamily P, member 2 (OR5P2); and
olfactory receptor, family 10, subfamily P, member 1 (OR10P1).
さらに、多能性幹細胞が以下に示す5個のケモカイン受容体の少なくとも一つが陽性の請求項1〜12のいずれか1項に記載の他家移植用細胞治療用組成物:
chemokine (C-C motif) receptor 5(CCR5);
chemokine (C-X-C motif) receptor 4(CXCR4);
chemokine (C-C motif) receptor 1(CCR1);
Duffy blood group, chemokine receptor(DARC);及び
chemokine (C-X-C motif) receptor 7(CXCR7)。
Furthermore, the composition for cell transplantation for allogeneic transplantation according to any one of claims 1 to 12, wherein at least one of the five chemokine receptors shown below is positive for pluripotent stem cells:
chemokine (CC motif) receptor 5 (CCR5);
chemokine (CXC motif) receptor 4 (CXCR4);
chemokine (CC motif) receptor 1 (CCR1);
Duffy blood group, chemokine receptor (DARC); and
chemokine (CXC motif) receptor 7 (CXCR7).
さらに、多能性幹細胞が中胚葉系組織または間葉系組織由来である、請求項1〜13のいずれか1項に記載の他家移植用細胞治療用組成物。   Furthermore, the composition for cell therapy for xenotransplantation of any one of Claims 1-13 whose pluripotent stem cell is a mesoderm type | system | group or a mesenchymal type | system | group origin.
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US20020156122A1 (en) * 2000-09-19 2002-10-24 Novlmmune S.A. Statins (HMG-CoA reductase inhibitors) as a novel type of immunomodulator, immunosuppressor and anti-inflammatory agent
US9550975B2 (en) * 2009-07-15 2017-01-24 Mari Dezawa SSEA-3 pluripotent stem cell isolated from body tissue

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* Cited by examiner, † Cited by third party
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