WO2010150650A1 - Novel method for maintaining stem cells in an undifferentiated state - Google Patents

Novel method for maintaining stem cells in an undifferentiated state Download PDF

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WO2010150650A1
WO2010150650A1 PCT/JP2010/059705 JP2010059705W WO2010150650A1 WO 2010150650 A1 WO2010150650 A1 WO 2010150650A1 JP 2010059705 W JP2010059705 W JP 2010059705W WO 2010150650 A1 WO2010150650 A1 WO 2010150650A1
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cells
cell
undifferentiated state
expression vector
maintaining
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雅規 本田
剛和 三上
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学校法人日本大学
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention uses CD271 (NGFR, nerve growth factor receptor: low affinity nerve growth factor receptor or p75NTR, p75 neurotrophin receptor: p75 nerve growth factor receptor, hereinafter referred to as CD271), mesenchymal stem cells, ES cells,
  • CD271 nerve growth factor receptor: low affinity nerve growth factor receptor or p75NTR, p75 neurotrophin receptor: p75 nerve growth factor receptor, hereinafter referred to as CD271
  • NGFR nerve growth factor receptor: low affinity nerve growth factor receptor or p75NTR
  • p75 neurotrophin receptor p75 nerve growth factor receptor
  • the present invention relates to a method for maintaining an undifferentiated state such as iPS cells.
  • MSCs mesenchymal stem cells
  • ES cells ES cells
  • iPS cells are undifferentiated and are known as pluripotent cells.
  • MSCs mesenchymal stem cells
  • ES cells ES cells
  • iPS cells are undifferentiated and are known as pluripotent cells.
  • methods for maintaining various undifferentiated states have been studied. For example, methods for introducing genes encoding Fibrillalin, AOF2, TIF1 ⁇ , etc. into these cells (Patent Documents 1 to 3), and proteins of the Wnt family
  • Patent Document 4 A method of culturing using a maintenance medium in an undifferentiated state containing lysine
  • Fibrillarin, AOF2, and TIF1 ⁇ are known as genes that are specifically expressed in mouse ES cells in the presence of LIF (leukemia inhibitor factor), which is necessary when mouse ES cells proliferate while maintaining an undifferentiated state. ing.
  • LIF leukemia inhibitor factor
  • genes are introduced, resistance genes for drugs such as geneticin and hygromycin are simultaneously introduced, and cells are selected using these drugs.
  • this method merely confirms the expression of the drug resistance gene, and does not directly confirm the expression of the target gene itself.
  • cells that have become drug resistant must be cultured from a single colony, but this requires long-term culture, There is also the possibility of affecting differentiation. Therefore, methods for introducing genes expressed in the cytoplasm or nucleus, such as Fibrillalin, AOF2, and TIF1 ⁇ , have various problems.
  • CD271 has been conventionally known to be localized and expressed in the cell membrane of cells of the nervous system and to be involved in the development, survival and differentiation of nerve cells. In recent years, it has been shown that it is specifically expressed in the cell membrane of MSCs, and a method for concentrating MSCs from a bone marrow extract using CD271 has been developed as an optimal marker for MSCs (Patent Literature). 5). Since CD271 is localized and expressed in the cell membrane, it differs from the case of introducing a gene expressed in the cytoplasm or nucleus such as Fibrillarin, AOF2, TIF1 ⁇ .
  • CD271 when CD271 is introduced, there is an advantage that cells expressing CD271 and non-expressing cells can be selected alive by using a fluorescently labeled antibody against CD271.
  • CD271 in MSCs has not been clarified, and it has not been predicted that the use of CD271 can maintain undifferentiated states such as mesenchymal stem cells, ES cells, and iPS cells.
  • An object of the present invention is to provide a method for maintaining an undifferentiated state of mesenchymal stem cells, ES cells, iPS cells and the like using CD271.
  • the present inventors introduce a vector that expresses CD271 into pluripotent undifferentiated cells such as mesenchymal stem cells, ES cells, and iPS cells.
  • pluripotent undifferentiated cells such as mesenchymal stem cells, ES cells, and iPS cells.
  • bone marrow-derived mesenchymal stem cells or dental pulp-derived mesenchymal stem cells that are forcibly expressed by introducing a vector that expresses CD271 can be used for osteoblasts, adipocytes, and the like. It was confirmed that differentiation and differentiation into osteoblasts / odontoblasts were suppressed.
  • the present invention relates to the following methods (1) to (9) for maintaining an undifferentiated state of mesenchymal stem cells, ES cells, iPS cells and the like.
  • (1) A method of maintaining an undifferentiated state of a cell by introducing a CD271 expression vector or a cell membrane permeabilizing recombinant CD271 protein into an undifferentiated state cell.
  • (2) The method according to (1) above, wherein the CD271 expression vector is an expression vector into which all or part of the gene encoding CD271 described in SEQ ID NO: 1 of the Sequence Listing is incorporated.
  • the undifferentiated cells are any of mesenchymal stem cells, ES cells, or iPS cells.
  • a cell in which an undifferentiated state is maintained by the method according to any one of (1) to (4) above.
  • a pharmaceutical for treating metabolic syndrome by inhibiting cell differentiation into adipocytes comprising the composition according to (8) above.
  • other preferred embodiments according to the present invention are as follows.
  • A) A method of suppressing cell differentiation into adipocytes or maintaining an undifferentiated state of cells by administering the composition according to (8) above.
  • B) A method for preventing or treating metabolic syndrome by suppressing cell differentiation into adipocytes with a pharmaceutical comprising the composition according to (8) above.
  • a vector that expresses CD271 and a cell membrane permeabilizing recombinant CD271 protein can also be provided as substances that maintain the undifferentiated state of these cells.
  • Example 2 It is the figure which showed CD271 expression vector (Example 1). It is the figure which confirmed the expression of CD271 in the CD271 positive cell (Test Example 1). It is the figure which showed the correlation of the activity level of the alkaline phosphatase in CD271 positive cell, and the expression level of CD271 (Test Example 2). (Example 2) which confirmed the maintenance of the undifferentiated state in the CD271 forced expression cell. (Example 2) which confirmed the maintenance of the undifferentiated state in the CD271 forced expression cell.
  • the “method for maintaining the undifferentiated state of a cell” of the present invention is achieved by introducing a recombinant gene containing a gene encoding CD271 or a CD271 expression vector into which the recombinant gene has been inserted into a cell such as a stem cell. Any method can be included as long as it can maintain the undifferentiated state.
  • the “method for maintaining the undifferentiated state of cells” of the present invention preferably controls the undifferentiated state of stem cells by forcibly expressing CD271 in the introduced cells such as stem cells by introducing a CD271 expression vector.
  • the “CD271 expression vector” of the present invention may include any recombinant gene containing a gene encoding CD271 or any vector capable of expressing CD271 into which the recombinant gene has been inserted.
  • Commercial products such as FCC117C02 (FIG. 1) can be mentioned.
  • examples include those in which a gene encoding CD271 is incorporated into an expression vector such as pcDNA3.1 (Invitrogen), pCMV5, pEGFP series (Clontech), p3XFLAG-CMV series (SIGMA).
  • the CD271 gene incorporated in the “CD271 expression vector” of the present invention may not be the entire gene encoding CD271, as long as it can maintain the undifferentiated state of cells such as stem cells, and the functional region of CD271, etc. It may be a gene encoding only a necessary sequence.
  • the “cell membrane permeabilizing recombinant CD271 protein” of the present invention refers to a CD271 protein to which a substance having cell membrane permeability such as polyarginine is bound.
  • the CD271 protein introduced into the stem cells or the like is not the entire CD271 protein as long as it can maintain the undifferentiated state of the cells such as the stem cells, but a part thereof is bound to a substance having cell membrane permeability such as polyarginine. It may be a thing.
  • the “undifferentiated state cell” of the present invention refers to a cell that maintains two functions of self-renewal ability and pluripotency, and in mesenchymal stem cells, ES cells, iPS cells, etc. A cell in which these functions are maintained.
  • the “kit for maintaining an undifferentiated state of a cell” of the present invention is a “CD271 expression vector” for expressing CD271 in a cell such as a stem cell or a “kit for introducing CD271 into a cell such as a stem cell”.
  • CD271 expression vector for expressing CD271 in a cell such as a stem cell
  • kit for introducing CD271 into a cell such as a stem cell “Cell membrane permeabilized recombinant CD271 protein” and a kit composed of a combination of one or more of a series of reagents necessary for the introduction thereof.
  • a composition containing CD271 as an active ingredient for inhibiting cell differentiation or maintaining the undifferentiated state of cells means that CD271 is used as an active ingredient to inhibit cell differentiation or maintain the undifferentiated state of cells. Any composition that can be used is included.
  • the CD271 used as an active ingredient may be the “CD271 expression vector” of the present invention for expressing CD271 in cells or the “cell membrane permeabilized recombinant CD271 protein” for introduction into cells.
  • This composition can also contain a pharmaceutically acceptable carrier in addition to CD271 as an active ingredient.
  • composition for suppressing cell differentiation or maintaining the undifferentiated state of cells is a composition capable of suppressing cell differentiation and maintaining the undifferentiated state of cells. Therefore, for example, by inhibiting the differentiation of cells into adipocytes, it can be used for the treatment of metabolic syndrome such as the manufacture of pharmaceuticals such as obesity inhibitors and the prevention and treatment of metabolic syndrome. In addition, by inhibiting the differentiation of cells into osteoblasts, it can also be used for the prevention and treatment of marbleosis and osteosclerosis caused by excessive bone formation, the production of pharmaceuticals therefor, and the like.
  • CD271 as an active ingredient in the present invention can be expected to function as a factor that suppresses hyperplasia caused by the proliferation of differentiated cells.
  • MSCs have pluripotency and differentiate into osteoblasts and adipocytes. Therefore, CD271 specifically expressed in MSCs is considered to have a function related to the multipotency of MSCs, and the difference in differentiation potential between cells expressing CD271 and cells not expressing CD271 was examined. .
  • the dental pulp cells having the ability to differentiate into osteoblasts / odontoblasts / adipocytes / neurons are mesenchymal cells and express CD90. Therefore, CD271 positive cells in dental pulp cells are It may correspond to leaf stem cells (MSCs).
  • MSCs leaf stem cells
  • Each of the sorted cells was cultured in ⁇ -MEM medium containing 20% FBS, 100 U / ml penicillin and 100 ⁇ g / ml streptomycin until it became confluent. Next, total RNA was produced from each cell, and based on this, mRNA expression of CD271 was confirmed using a real-time PCR method. As a result, it was confirmed that CD271 positive cells expressed CD271 about 40 times as compared with CD271 negative cells (FIG. 2).
  • Primer Perfect Real Time Primer
  • primer set ID HA036699
  • Takara Bio Reagents SYBR Premix Ex Taq II
  • Product Code RR081A
  • Analytical Equipment Smart Cycler II System C, System C
  • Test Example 2 CD271 positive cells and CD271 negative cells selected and cultured in the same manner as in Test Example 1 were mixed with osteoblast differentiation induction medium (100 ng / ml BMP-2, 50 ⁇ g / ml L-ascorbate phosphate, 10 mM ⁇ -glycophosphate, 20% FBS, ( ⁇ -MEM medium containing 100 U / ml penicillin, 100 ⁇ g / ml streptomycin) for 6 days, 12 days, and 24 days, and the activity of alkaline phosphatase, which is an index of osteoblast differentiation, was determined to be alkaline phosphatase (ALP) staining solution.
  • osteoblast differentiation induction medium 100 ng / ml BMP-2, 50 ⁇ g / ml L-ascorbate phosphate, 10 mM ⁇ -glycophosphate, 20% FBS, ( ⁇ -MEM medium containing 100 U / ml penicillin, 100 ⁇ g /
  • ⁇ Method for maintaining the undifferentiated state of mesenchymal stem cells, ES cells, and iPS cells By introducing CD271 into a C3H10T1 / 2 cell, which is an undifferentiated mesenchymal stem cell line derived from a mouse, by constantly expressing CD271, the undifferentiated state of this cell was maintained.
  • CD271 expression vector into C3H10T1 / 2 cells
  • C3H10T1 / 2 cells were made 50% confluent in cell culture medium containing 10% fetal bovine serum, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin ( ⁇ -MEM). Pre-cultured in 6 well dishes until complete. Thereafter, the culture solution was replaced with a cell culture solution ( ⁇ -MEM) containing 5% fetal bovine serum, and pre-culture was performed for 2 to 3 hours.
  • a C271 expression vector (TOYOBO cDNA Clones NGFR, Code No. FCC117C02) purchased from TOYOBO was cleaved with DNA restriction enzyme ScaI to generate a linearized vector.
  • the geneticin resistance gene expression region contained in the pcDNA3.1 vector was excised with DNA restriction enzymes KpnI and ApLI.
  • a linearized CD271 expression vector (2 ⁇ g) and a DNA (0.3 ⁇ g) containing a geneticin resistance gene expression region excised from the pcDNA3.1 vector were transfected into cells using lipofectamine LTX of Invitrogen according to the attached manual.
  • the cell culture medium was replaced with a cell culture medium containing 10% fetal bovine serum, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin, and cultured for 24 hours. Thereafter, the cells were cultured for 10 days in the presence of geneticin. Thereafter, FACS analysis was performed using a fluorescently labeled CD271 antibody, and cells expressing CD271 were selected.
  • Example 1 Induction of differentiation into osteoblasts In the ⁇ -MEM medium containing 10% FBS, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin, until the C3H10T1 / 2 cells of Example 1 (CD271 forced expression cells) became confluent. Cultured for about 3 weeks.
  • osteoblast differentiation medium 100 ng / ml BMP-2, 10-8M Dexamethasone, 50 ⁇ g / ml L-ascorbate phosphate, 10 mM ⁇ -glycophosphate, 10% FBS, 100 U / ml penicillin, ⁇ containing 100 ⁇ g / ml streptomycin
  • -MEM medium was further cultured for 12 days, and alkaline phosphatase staining was performed in the same manner as in Test Example 2 above.
  • cells into which only an expression vector not containing CD271 was introduced were used.
  • Example 2 Induction of differentiation into adipocytes
  • the C3H10T1 / 2 cells (CD271 forced expression cells) of Example 1 were cultured until confluent as in 1) above, and then adipocyte differentiation induction medium (100 ng / ml BMP- 2, 10-8 M Dexamethasone, ⁇ -MEM medium containing 100% FBS, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin) for 12 days, and formation of fat globules as an indicator of fat cells Observed by staining. Cell nuclei were stained with hematoxylin. For comparison, cells into which only an expression vector not containing CD271 was introduced (control cells) were used.
  • adipocyte differentiation induction medium 100 ng / ml BMP- 2, 10-8 M Dexamethasone, ⁇ -MEM medium containing 100% FBS, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin
  • a vector that expresses CD271 and a cell membrane permeabilizing recombinant CD271 protein can also be provided as substances that maintain the undifferentiated state of these cells.
  • a composition for suppressing cell differentiation and maintaining an undifferentiated state containing CD271 as an active ingredient to provide a pharmaceutical (eg, a pharmaceutical for treating metabolic syndrome by inhibiting adipocyte differentiation). You can also.

Abstract

Disclosed is a method for maintaining the undifferentiated state of mesenchymal stem cells, ES cells, and iPS cells using CD271. By inserting a vector expressing CD271, or by directly inserting cell membrane-permeabilized recombinant CD271 protein into pluripotent undifferentiated cells such as mesenchymal stem cells, ES cells, and iPS cells, the undifferentiated state of these cells is maintained.

Description

幹細胞の未分化状態を維持する新規方法A novel method for maintaining the undifferentiated state of stem cells
　本発明は、ＣＤ２７１（ＮＧＦＲ，ｎｅｒｖｅ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ　ｒｅｃｅｐｔｏｒ：低親和性神経成長因子レセプターまたはｐ７５ＮＴＲ、ｐ７５　ｎｅｕｒｏｔｒｏｐｈｉｎ　ｒｅｃｅｐｔｏｒ：ｐ７５神経成長因子レセプター、以下、ＣＤ２７１とする）を用いて間葉系幹細胞、ＥＳ細胞、ｉＰＳ細胞などの未分化状態を維持する方法に関する。 The present invention uses CD271 (NGFR, nerve growth factor receptor: low affinity nerve growth factor receptor or p75NTR, p75 neurotrophin receptor: p75 nerve growth factor receptor, hereinafter referred to as CD271), mesenchymal stem cells, ES cells, The present invention relates to a method for maintaining an undifferentiated state such as iPS cells.
　間葉系幹細胞（以下、ＭＳＣｓとする）、ＥＳ細胞、ｉＰＳ細胞などの細胞は、未分化であり、多能性を有する細胞として知られている。しかし、長期培養や培養条件の微細な変化によって多能性が喪失されたり、非特異的な分化が誘発されたりするという問題があった。
　そのため、様々な未分化状態を維持する方法が検討されており、例えば、Ｆｉｂｒｉｌｌａｒｉｎ、ＡＯＦ２、ＴＩＦ１β等をコードする遺伝子をこれらの細胞に導入する方法（特許文献１～３）や、Ｗｎｔファミリーのタンパク質を含む未分化状態の維持用培地を用いて培養する方法（特許文献４）等が開発されている。 Cells such as mesenchymal stem cells (hereinafter referred to as MSCs), ES cells, and iPS cells are undifferentiated and are known as pluripotent cells. However, there has been a problem that pluripotency is lost or nonspecific differentiation is induced by long-term culture or minute changes in culture conditions.
Therefore, methods for maintaining various undifferentiated states have been studied. For example, methods for introducing genes encoding Fibrillalin, AOF2, TIF1β, etc. into these cells (Patent Documents 1 to 3), and proteins of the Wnt family A method of culturing using a maintenance medium in an undifferentiated state containing lysine (Patent Document 4) has been developed.
　Ｆｉｂｒｉｌｌａｒｉｎ、ＡＯＦ２、ＴＩＦ１βは、マウスＥＳ細胞が未分化状態を維持しながら増殖する際に必要となるＬＩＦ（ｌｅｕｋｅｍｉａ　ｉｎｈｉｂｉｔｏｒ　ｆａｃｔｏｒ）存在下で、マウスＥＳ細胞に特異的に発現している遺伝子として知られている。しかし、これらは細胞質あるいは核内において発現するため、細胞が生きたままの状態で、目的とするタンパク質が発現したか否かを確認することは困難である。従って、タンパク質を発現している細胞と発現していない細胞を選別することが容易ではなく、導入処理後の細胞集団において、それぞれの細胞が混在した状態にあるため、その後の実験、解析等の障害となる可能性がある。
　通常、この問題を解決するために、遺伝子導入を行う際に、ジェネティシン、ハイグロマイシン等の薬剤に対する耐性遺伝子を同時に導入し、それらの薬剤を用いて細胞を選別する。しかし、この方法はあくまでも薬剤耐性遺伝子の発現を確認するものであって、目的遺伝子の発現そのものを直接的に確認するものではない。また、目的遺伝子を発現している細胞のみを選別するためには、薬剤耐性となった細胞をシングルコロニーから培養する必要があるが、これには長期間の培養が必要となるため、その後の分化に影響を与える可能性も考えられる。従って、Ｆｉｂｒｉｌｌａｒｉｎ、ＡＯＦ２、ＴＩＦ１β等の細胞質あるいは核内において発現する遺伝子を導入する方法はさまざまな問題を含んでいる。 Fibrillarin, AOF2, and TIF1β are known as genes that are specifically expressed in mouse ES cells in the presence of LIF (leukemia inhibitor factor), which is necessary when mouse ES cells proliferate while maintaining an undifferentiated state. ing. However, since these are expressed in the cytoplasm or nucleus, it is difficult to confirm whether or not the target protein is expressed while the cells remain alive. Therefore, it is not easy to select cells that express protein and cells that do not express, and in the cell population after the introduction treatment, each cell is in a mixed state. It can be an obstacle.
Usually, in order to solve this problem, when genes are introduced, resistance genes for drugs such as geneticin and hygromycin are simultaneously introduced, and cells are selected using these drugs. However, this method merely confirms the expression of the drug resistance gene, and does not directly confirm the expression of the target gene itself. In addition, in order to select only cells expressing the target gene, cells that have become drug resistant must be cultured from a single colony, but this requires long-term culture, There is also the possibility of affecting differentiation. Therefore, methods for introducing genes expressed in the cytoplasm or nucleus, such as Fibrillalin, AOF2, and TIF1β, have various problems.
　ＣＤ２７１は、従来、神経系の細胞の細胞膜に局在して発現し、神経細胞の発生、生存および分化に関与することが知られていた。そして、近年、ＭＳＣｓの細胞膜にも特異的に発現していることが示され、ＭＳＣｓの最適なマーカーとして、ＣＤ２７１を用いた骨髄抽出液などからＭＳＣｓを濃縮する方法が開発されている（特許文献５）。
　ＣＤ２７１は細胞膜に局在して発現することから、Ｆｉｂｒｉｌｌａｒｉｎ、ＡＯＦ２、ＴＩＦ１β等の細胞質あるいは核内において発現する遺伝子を導入する場合と異なる。そのため、ＣＤ２７１を導入した場合に、蛍光ラベルしたＣＤ２７１に対する抗体を用いることによって、ＣＤ２７１を発現している細胞と発現していない細胞を生きたまま選別することが可能であるという利点がある。
　しかし、ＣＤ２７１のＭＳＣｓにおける機能については明らかにされておらず、ＣＤ２７１を用いることにより、間葉系幹細胞、ＥＳ細胞、ｉＰＳ細胞などの未分化状態が維持できることも予測されていなかった。 CD271 has been conventionally known to be localized and expressed in the cell membrane of cells of the nervous system and to be involved in the development, survival and differentiation of nerve cells. In recent years, it has been shown that it is specifically expressed in the cell membrane of MSCs, and a method for concentrating MSCs from a bone marrow extract using CD271 has been developed as an optimal marker for MSCs (Patent Literature). 5).
Since CD271 is localized and expressed in the cell membrane, it differs from the case of introducing a gene expressed in the cytoplasm or nucleus such as Fibrillarin, AOF2, TIF1β. Therefore, when CD271 is introduced, there is an advantage that cells expressing CD271 and non-expressing cells can be selected alive by using a fluorescently labeled antibody against CD271.
However, the function of CD271 in MSCs has not been clarified, and it has not been predicted that the use of CD271 can maintain undifferentiated states such as mesenchymal stem cells, ES cells, and iPS cells.
特開２００９－７２１８６号公報JP 2009-72186 A 特開２００９－４５００４号公報JP 2009-45004 A 特開２００９－１１２５５号公報JP 2009-11255 A 特開２００６－３４５７０２号公報JP 2006-345702 A 特開２００９－６０８４０号公報JP 2009-60840 A
　本発明は、ＣＤ２７１を用いて間葉系幹細胞、ＥＳ細胞、ｉＰＳ細胞などの未分化状態を維持する方法の提供を課題とする。 An object of the present invention is to provide a method for maintaining an undifferentiated state of mesenchymal stem cells, ES cells, iPS cells and the like using CD271.
　本発明者らは、上記課題を解決するために鋭意研究を行った結果、間葉系幹細胞、ＥＳ細胞、ｉＰＳ細胞などの多能性の未分化細胞に、ＣＤ２７１を発現するベクターを導入する、または細胞膜透過化型リコンビナントＣＤ２７１タンパク質を直接導入することによって、これらの細胞の各種細胞系統への分化が抑制され、これらの細胞の未分化状態を維持できることを見出し、本発明を完成するに至った。
　本発明者らは、本発明において、ＣＤ２７１を発現するベクターを導入することで強制発現させた骨髄由来の間葉系幹細胞や歯髄由来の間葉系幹細胞において、骨芽細胞および脂肪細胞等への分化や、骨芽細胞／象牙芽細胞への分化が抑制されたことを確認している。 As a result of intensive studies to solve the above problems, the present inventors introduce a vector that expresses CD271 into pluripotent undifferentiated cells such as mesenchymal stem cells, ES cells, and iPS cells. Alternatively, by directly introducing the cell membrane permeabilizing recombinant CD271 protein, it was found that the differentiation of these cells into various cell lines can be suppressed and the undifferentiated state of these cells can be maintained, and the present invention has been completed. .
In the present invention, in the present invention, bone marrow-derived mesenchymal stem cells or dental pulp-derived mesenchymal stem cells that are forcibly expressed by introducing a vector that expresses CD271 can be used for osteoblasts, adipocytes, and the like. It was confirmed that differentiation and differentiation into osteoblasts / odontoblasts were suppressed.
　すなわち、本発明は次の（１）～（９）の間葉系幹細胞、ＥＳ細胞、ｉＰＳ細胞などの未分化状態を維持する方法等に関する。
（１）未分化状態の細胞にＣＤ２７１発現ベクターまたは細胞膜透過化型リコンビナントＣＤ２７１タンパク質を導入することによって、細胞の未分化状態を維持する方法。
（２）ＣＤ２７１発現ベクターが配列表配列番号１に記載のＣＤ２７１をコードする遺伝子の全部または一部が組み込まれた発現ベクターである上記（１）に記載の方法。
（３）ＣＤ２７１発現ベクターが図１に記載のＣＤ２７１発現ベクターである上記（１）または（２）に記載の方法。
（４）未分化状態の細胞が、間葉系幹細胞、ＥＳ細胞またはｉＰＳ細胞のいずれかである上記（１）～（３）のいずれかに記載の方法。
（５）上記（１）～（４）のいずれかに記載の方法によって、未分化状態が維持された細胞。
（６）細胞の未分化状態を維持するためのＣＤ２７１発現ベクターまたは細胞膜透過化型リコンビナントＣＤ２７１タンパク質。
（７）ＣＤ２７１発現ベクターまたは細胞膜透過化型リコンビナントＣＤ２７１タンパク質のいずれか１つ以上を含む細胞の未分化状態を維持するためのキット。
（８）ＣＤ２７１を有効成分とする、細胞分化を抑制する、または細胞の未分化状態を維持するための組成物。
（９）上記（８）に記載の組成物を含む、脂肪細胞への細胞分化を抑制することによるメタボリックシンドローム対処用の医薬品。
　また、本発明に係る他の好ましい態様は以下の通りである。
（ａ）上記（８）に記載の組成物を投与することにより、脂肪細胞への細胞分化を抑制する、または細胞の未分化状態を維持する方法。
（ｂ）上記（８）に記載の組成物を含む医薬品により、脂肪細胞への細胞分化を抑制することによるメタボリックシンドロームの予防または治療方法。 That is, the present invention relates to the following methods (1) to (9) for maintaining an undifferentiated state of mesenchymal stem cells, ES cells, iPS cells and the like.
(1) A method of maintaining an undifferentiated state of a cell by introducing a CD271 expression vector or a cell membrane permeabilizing recombinant CD271 protein into an undifferentiated state cell.
(2) The method according to (1) above, wherein the CD271 expression vector is an expression vector into which all or part of the gene encoding CD271 described in SEQ ID NO: 1 of the Sequence Listing is incorporated.
(3) The method according to (1) or (2) above, wherein the CD271 expression vector is the CD271 expression vector described in FIG.
(4) The method according to any one of (1) to (3) above, wherein the undifferentiated cells are any of mesenchymal stem cells, ES cells, or iPS cells.
(5) A cell in which an undifferentiated state is maintained by the method according to any one of (1) to (4) above.
(6) A CD271 expression vector or a cell membrane permeabilizing recombinant CD271 protein for maintaining the undifferentiated state of cells.
(7) A kit for maintaining an undifferentiated state of a cell containing any one or more of a CD271 expression vector and a cell membrane permeabilizing recombinant CD271 protein.
(8) A composition containing CD271 as an active ingredient, for suppressing cell differentiation or maintaining an undifferentiated state of cells.
(9) A pharmaceutical for treating metabolic syndrome by inhibiting cell differentiation into adipocytes, comprising the composition according to (8) above.
Further, other preferred embodiments according to the present invention are as follows.
(A) A method of suppressing cell differentiation into adipocytes or maintaining an undifferentiated state of cells by administering the composition according to (8) above.
(B) A method for preventing or treating metabolic syndrome by suppressing cell differentiation into adipocytes with a pharmaceutical comprising the composition according to (8) above.
　本発明の方法を用いることにより、未分化状態が長期的に維持された間葉系幹細胞、ＥＳ細胞、ｉＰＳ細胞を提供することができる。また、ＣＤ２７１を発現するベクターや、細胞膜透過化型リコンビナントＣＤ２７１タンパク質を、これらの細胞の未分化状態維持物質として提供することもできる。 By using the method of the present invention, it is possible to provide mesenchymal stem cells, ES cells, and iPS cells whose undifferentiated state has been maintained for a long time. Moreover, a vector that expresses CD271 and a cell membrane permeabilizing recombinant CD271 protein can also be provided as substances that maintain the undifferentiated state of these cells.
ＣＤ２７１発現ベクターを示した図である（実施例１）。It is the figure which showed CD271 expression vector (Example 1). ＣＤ２７１ポジティブ細胞におけるＣＤ２７１の発現を確認した図である（試験例１）。It is the figure which confirmed the expression of CD271 in the CD271 positive cell (Test Example 1). ＣＤ２７１ポジティブ細胞におけるアルカリフォスファターゼの活性レベルとＣＤ２７１の発現量の相関を示した図である（試験例２）。It is the figure which showed the correlation of the activity level of the alkaline phosphatase in CD271 positive cell, and the expression level of CD271 (Test Example 2). ＣＤ２７１強制発現細胞における未分化状態の維持を確認した図である（実施例２）。(Example 2) which confirmed the maintenance of the undifferentiated state in the CD271 forced expression cell. ＣＤ２７１強制発現細胞における未分化状態の維持を確認した図である（実施例２）。(Example 2) which confirmed the maintenance of the undifferentiated state in the CD271 forced expression cell.
　本発明の「細胞の未分化状態を維持する方法」は、ＣＤ２７１をコードする遺伝子を含む組換え遺伝子又は当該組換え遺伝子が挿入されたＣＤ２７１発現ベクターを幹細胞等の細胞に導入することで、細胞の未分化状態を維持することができる方法であればいずれの方法も含むことができる。本発明の「細胞の未分化状態を維持する方法」は、ＣＤ２７１発現ベクターの導入により、幹細胞等の導入細胞内でＣＤ２７１を強制発現させることで幹細胞の未分化状態を制御することが好ましい。 The “method for maintaining the undifferentiated state of a cell” of the present invention is achieved by introducing a recombinant gene containing a gene encoding CD271 or a CD271 expression vector into which the recombinant gene has been inserted into a cell such as a stem cell. Any method can be included as long as it can maintain the undifferentiated state. The “method for maintaining the undifferentiated state of cells” of the present invention preferably controls the undifferentiated state of stem cells by forcibly expressing CD271 in the introduced cells such as stem cells by introducing a CD271 expression vector.
　本発明の「ＣＤ２７１発現ベクター」は、ＣＤ２７１をコードする遺伝子を含む組換え遺伝子又は当該組換え遺伝子が挿入されたＣＤ２７１を発現できるベクターであればいずれのものも含むことができる。例えば、配列表配列番号１に記載のＣＤ２７１をコードする遺伝子が配列表配列番号２に記載の発現ベクターｐＭＥ１８ＳＦＬ３に組み込まれたＴＯＹＯＢＯ　ｃＤＮＡ　Ｃｌｏｎｅｓ　ＮＧＦＲ，Ｃｏｄｅ　Ｎｏ．ＦＣＣ１１７Ｃ０２（図１）等の市販の製品が挙げられる。また、ＣＤ２７１をコードする遺伝子をｐｃＤＮＡ３．１（インビトロジェン社）、ｐＣＭＶ５、ｐＥＧＦＰシリーズ（クロンテック社）、ｐ３ＸＦＬＡＧ－ＣＭＶシリーズ（ＳＩＧＭＡ社）等の発現ベクターに組み込んだもの等が挙げられる。本発明の「ＣＤ２７１発現ベクター」に組み込まれるＣＤ２７１遺伝子は、幹細胞等の細胞の未分化状態を維持できるものであれば、ＣＤ２７１をコードする遺伝子の全部でなくてもよく、ＣＤ２７１の機能領域等、必要な配列のみをコードする遺伝子であってもよい。 The “CD271 expression vector” of the present invention may include any recombinant gene containing a gene encoding CD271 or any vector capable of expressing CD271 into which the recombinant gene has been inserted. For example, a TOYOBO cDNA Clones NGFR, Code No. 2 in which a gene encoding CD271 described in Sequence Listing SEQ ID NO: 1 is incorporated into the expression vector pME18SFL3 described in Sequence Listing SEQ ID NO: 2. Commercial products such as FCC117C02 (FIG. 1) can be mentioned. Further, examples include those in which a gene encoding CD271 is incorporated into an expression vector such as pcDNA3.1 (Invitrogen), pCMV5, pEGFP series (Clontech), p3XFLAG-CMV series (SIGMA). The CD271 gene incorporated in the “CD271 expression vector” of the present invention may not be the entire gene encoding CD271, as long as it can maintain the undifferentiated state of cells such as stem cells, and the functional region of CD271, etc. It may be a gene encoding only a necessary sequence.
　本発明の「細胞膜透過化型リコンビナントＣＤ２７１タンパク質」とは、ポリアルギニンなどの細胞膜透過性を有する物質が結合されたＣＤ２７１タンパク質のことをいう。ここで、幹細胞等に導入するＣＤ２７１タンパク質は、幹細胞等の細胞の未分化状態を維持できるものであればＣＤ２７１タンパク質全部でなく、その一部をポリアルギニンなどの細胞膜透過性を有する物質に結合したものであってもよい。 The “cell membrane permeabilizing recombinant CD271 protein” of the present invention refers to a CD271 protein to which a substance having cell membrane permeability such as polyarginine is bound. Here, the CD271 protein introduced into the stem cells or the like is not the entire CD271 protein as long as it can maintain the undifferentiated state of the cells such as the stem cells, but a part thereof is bound to a substance having cell membrane permeability such as polyarginine. It may be a thing.
　本発明の「未分化状態の細胞」とは、自己複製能と多分化能の二つの機能を維持している状態の細胞のことをいい、間葉系幹細胞、ＥＳ細胞またはｉＰＳ細胞等において、これらの機能が維持された細胞のことをいう。 The “undifferentiated state cell” of the present invention refers to a cell that maintains two functions of self-renewal ability and pluripotency, and in mesenchymal stem cells, ES cells, iPS cells, etc. A cell in which these functions are maintained.
　本発明の「細胞の未分化状態を維持するためのキット」とは、ＣＤ２７１を幹細胞等の細胞内で発現させるための「ＣＤ２７１発現ベクター」やＣＤ２７１を幹細胞等の細胞内に導入するための「細胞膜透過化型リコンビナントＣＤ２７１タンパク質」、さらに、これらの導入等に必要な一連の試薬一式等を１つ以上組合せてキット化したもののことをいう。 The “kit for maintaining an undifferentiated state of a cell” of the present invention is a “CD271 expression vector” for expressing CD271 in a cell such as a stem cell or a “kit for introducing CD271 into a cell such as a stem cell”. “Cell membrane permeabilized recombinant CD271 protein” and a kit composed of a combination of one or more of a series of reagents necessary for the introduction thereof.
　「ＣＤ２７１を有効成分とする、細胞分化を抑制する、または細胞の未分化状態を維持するための組成物」とは、ＣＤ２７１を有効成分として、細胞分化の抑制や、細胞の未分化状態を維持できる組成物であればいずれのものも含まれる。
　有効成分とするＣＤ２７１は、ＣＤ２７１を細胞内で発現するための本発明の「ＣＤ２７１発現ベクター」や、細胞内に導入するための「細胞膜透過化型リコンビナントＣＤ２７１タンパク質」であってもよい。この組成物は、有効成分とするＣＤ２７１以外に、薬学的に許容可能な担体等を含むこともできる。 “A composition containing CD271 as an active ingredient for inhibiting cell differentiation or maintaining the undifferentiated state of cells” means that CD271 is used as an active ingredient to inhibit cell differentiation or maintain the undifferentiated state of cells. Any composition that can be used is included.
The CD271 used as an active ingredient may be the “CD271 expression vector” of the present invention for expressing CD271 in cells or the “cell membrane permeabilized recombinant CD271 protein” for introduction into cells. This composition can also contain a pharmaceutically acceptable carrier in addition to CD271 as an active ingredient.
　本発明の「ＣＤ２７１を有効成分とする、細胞分化を抑制する、または細胞の未分化状態を維持するための組成物」は、細胞分化の抑制や、細胞の未分化状態が維持できる組成物であることから、例えば、細胞の脂肪細胞への分化を抑制することにより、肥満抑制剤等の医薬品の製造や、メタボリックシンドロームの予防や治療等、メタボリックシンドロームの対処に用いることができる。また、細胞の骨芽細胞への分化を抑制することにより、過剰な骨形成によって生じる大理石症、骨硬化症などの予防や治療、そのための医薬品の製造等に用いることもできる。本発明において有効成分とされるＣＤ２７１は、分化した細胞の増殖によって起きる過形成を抑制する因子として機能することが期待できる。 The “composition for suppressing cell differentiation or maintaining the undifferentiated state of cells” comprising CD271 as an active ingredient of the present invention is a composition capable of suppressing cell differentiation and maintaining the undifferentiated state of cells. Therefore, for example, by inhibiting the differentiation of cells into adipocytes, it can be used for the treatment of metabolic syndrome such as the manufacture of pharmaceuticals such as obesity inhibitors and the prevention and treatment of metabolic syndrome. In addition, by inhibiting the differentiation of cells into osteoblasts, it can also be used for the prevention and treatment of marbleosis and osteosclerosis caused by excessive bone formation, the production of pharmaceuticals therefor, and the like. CD271 as an active ingredient in the present invention can be expected to function as a factor that suppresses hyperplasia caused by the proliferation of differentiated cells.
　以下、試験例、実施例をあげて本発明をさらに詳細に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to test examples and examples, but the present invention is not limited thereto.
［試験例］
　ＭＳＣｓの最も顕著な特徴は多分化能を有し、骨芽細胞や脂肪細胞などに分化することである。そこで、ＭＳＣｓに特異的に発現するＣＤ２７１は、このＭＳＣｓの多分化能に関与した機能を有すると考え、ＣＤ２７１を発現している細胞と発現していない細胞において、その分化能の違いを検討した。 [Test example]
The most prominent feature of MSCs is that they have pluripotency and differentiate into osteoblasts and adipocytes. Therefore, CD271 specifically expressed in MSCs is considered to have a function related to the multipotency of MSCs, and the difference in differentiation potential between cells expressing CD271 and cells not expressing CD271 was examined. .
［試験例１］
　フローサイトメトリーを用いて、ヒト乳歯歯髄からＣＤ２７１を発現している細胞（ＣＤ２７１ポジティブ細胞）とＣＤ２７１を発現していない細胞（ＣＤ２７１ネガティブ細胞）をそれぞれフローサイトメトリー（ＣＤ２７１：ＰＥ－Ｍｏｕｓｅ　Ａｎｔｉ－Ｈｕｍａｎ　ＣＤ２７１　Ｃａｔ：５５７１９６，ＢＤ　ＰｈａｒｍｉｎｇｅｎＴＭ、ＡＰＣ　Ｍｏｕｓｅ－Ａｎｔｉ－Ｈｕｍａｎ　ＣＤ９０　Ｃａｔ：５５９８６９，ＢＤ　ＰｈａｒｍｉｎｇｅｎＴＭ）を用いて選別した。
　まず、間葉系の幹細胞（ＭＳＣｓ）のマーカーであるＣＤ９０の抗体を用いて間葉系の幹細胞（ＭＳＣｓ）を単離し、次にＣＤ２７１の抗体を用いてこのうちのＣＤ２７１ポジティブ細胞を選別した。骨芽細胞／象牙芽細胞・脂肪細胞・神経細胞への分化能を有している歯髄細胞は間葉系細胞であり、ＣＤ９０を発現していることから、歯髄細胞におけるＣＤ２７１ポジティブ細胞も、間葉系の幹細胞（ＭＳＣｓ）に該当し得る。
　選別した細胞をそれぞれ、２０％ＦＢＳ、１００Ｕ／ｍｌ　ｐｅｎｉｃｉｌｌｉｎ、１００μｇ／ｍｌ　ｓｔｒｅｐｔｏｍｙｃｉｎを含むα－ＭＥＭ培地でコンフルエント状態になるまで培養した。次にそれぞれの細胞からｔｏｔａｌ　ＲＮＡを生成し、これをもとにＣＤ２７１のｍＲＮＡ発現を、リアルタイムＰＣＲ法を用いて確認した。
　その結果、ＣＤ２７１ポジティブ細胞ではＣＤ２７１ネガティブ細胞と比較して約４０倍のＣＤ２７１を発現していることが確認された（図２）。
　リアルタイムＰＣＲ法には、以下のプライマー、試薬、解析機器を用いた。Ｐｒｉｍｅｒ：Ｐｅｒｆｅｃｔ　Ｒｅａｌ　Ｔｉｍｅ　Ｐｒｉｍｅｒ，ｐｒｉｍｅｒ　ｓｅｔ　ＩＤ：ＨＡ０３６６９９，タカラバイオ社試薬：ＳＹＢＲ　Ｐｒｅｍｉｘ　Ｅｘ　Ｔａｑ　ＩＩ，製品コード：ＲＲ０８１Ａ，タカラバイオ社解析機器：Ｓｍａｒｔ　Ｃｙｃｌｅｒ　ＩＩ　Ｓｙｓｔｅｍ，製品コード：ＳＣ２００Ｎ，Ｃｅｐｈｅｉｄ社 [Test Example 1]
Using flow cytometry, cells expressing CD271 (CD271 positive cells) and cells not expressing CD271 (CD271 negative cells) from human deciduous dental pulp were each flow cytometry (CD271: PE-Mouse Anti-Human). CD271 Cat: 557196, BD Pharmingen ™ , APC Mouse-Anti-Human CD90 Cat: 559869, BD Pharmingen ™ ).
First, mesenchymal stem cells (MSCs) were isolated using an antibody of CD90 which is a marker of mesenchymal stem cells (MSCs), and then CD271 positive cells were selected using an antibody of CD271. The dental pulp cells having the ability to differentiate into osteoblasts / odontoblasts / adipocytes / neurons are mesenchymal cells and express CD90. Therefore, CD271 positive cells in dental pulp cells are It may correspond to leaf stem cells (MSCs).
Each of the sorted cells was cultured in α-MEM medium containing 20% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin until it became confluent. Next, total RNA was produced from each cell, and based on this, mRNA expression of CD271 was confirmed using a real-time PCR method.
As a result, it was confirmed that CD271 positive cells expressed CD271 about 40 times as compared with CD271 negative cells (FIG. 2).
The following primers, reagents, and analysis equipment were used for the real-time PCR method. Primer: Perfect Real Time Primer, primer set ID: HA036699, Takara Bio Reagents: SYBR Premix Ex Taq II, Product Code: RR081A, Takara Bio Inc. Analytical Equipment: Smart Cycler II System C, System C
［試験例２］
　上記試験例１と同様に選別培養したＣＤ２７１ポジティブ細胞とＣＤ２７１ネガティブ細胞を、骨芽細胞分化誘導培地（１００ｎｇ／ｍｌ　ＢＭＰ－２、５０μｇ／ｍｌ　Ｌ－ａｓｃｏｒｂａｔｅ　ｐｈｏｓｐｈａｔｅ、１０ｍＭ　β－ｇｌｙｃｅｒｏｐｈｏｓｐｈａｔｅ、２０％　ＦＢＳ、１００Ｕ／ｍｌ　ｐｅｎｉｃｉｌｌｉｎ、１００μｇ／ｍｌ　ｓｔｒｅｐｔｏｍｙｃｉｎを含むα－ＭＥＭ培地）でさらに６日、１２日、２４日間培養し、骨芽細胞分化の指標とされるアルカリフォスファターゼの活性をａｌｋａｌｉｎｅ　ｐｈｏｓｐｈａｔａｓｅ　（ＡＬＰ）ｓｔａｉｎｉｎｇ　ｓｏｌｕｔｉｏｎ（ｒｅａｄｙ－ｔｏｕｓｅｔａｂｌｅｔｓ，Ｒｏｃｈｅ）を用いた染色によって比較した。また、上記試験例１と同様の方法で、培養したそれぞれの細胞からｔｏｔａｌＲＮＡを生成し、これをもとにＣＤ２７１のｍＲＮＡ発現を、リアルタイムＰＣＲ法を用いて確認した。
　その結果、ＣＤ２７１ポジティブ細胞では、ＣＤ２７１ネガティブ細胞と比較して、アルカリフォスファターゼの活性化が抑制された。また、ＣＤ２７１ポジティブ細胞においては、アルカリフォスファターゼの活性レベルの上昇にともない、ＣＤ２７１の発現量が減少した（図３）。 [Test Example 2]
CD271 positive cells and CD271 negative cells selected and cultured in the same manner as in Test Example 1 were mixed with osteoblast differentiation induction medium (100 ng / ml BMP-2, 50 μg / ml L-ascorbate phosphate, 10 mM β-glycophosphate, 20% FBS, (Α-MEM medium containing 100 U / ml penicillin, 100 μg / ml streptomycin) for 6 days, 12 days, and 24 days, and the activity of alkaline phosphatase, which is an index of osteoblast differentiation, was determined to be alkaline phosphatase (ALP) staining solution. Comparison was made by staining with (ready-tousetables, Roche). Further, total RNA was generated from each cultured cell by the same method as in Test Example 1 above, and based on this, mRNA expression of CD271 was confirmed using a real-time PCR method.
As a result, activation of alkaline phosphatase was suppressed in CD271 positive cells compared to CD271 negative cells. In addition, in CD271 positive cells, the expression level of CD271 decreased as the alkaline phosphatase activity level increased (FIG. 3).
　上記試験例１、２の結果より、ＣＤ２７１を発現しているヒト乳歯歯髄において、骨芽細胞への分化が抑制される傾向が観察されたことから、ＭＳＣｓに特異的に発現するＣＤ２７１が、細胞の分化を抑制する働きあることが示唆された。 From the results of Test Examples 1 and 2 above, it was observed that human deciduous dental pulp expressing CD271 showed a tendency to suppress differentiation into osteoblasts. Therefore, CD271 specifically expressed in MSCs is a cell. It was suggested that it has a function of suppressing the differentiation.
＜間葉系幹細胞、ＥＳ細胞、ｉＰＳ細胞の未分化状態を維持する方法＞
　マウス由来の未分化間葉系幹細胞株であるＣ３Ｈ１０Ｔ１／２細胞にＣＤ２７１を遺伝子導入することによって、恒常的にＣＤ２７１を発現させることで、この細胞の未分化状態を維持した。 <Method for maintaining the undifferentiated state of mesenchymal stem cells, ES cells, and iPS cells>
By introducing CD271 into a C3H10T1 / 2 cell, which is an undifferentiated mesenchymal stem cell line derived from a mouse, by constantly expressing CD271, the undifferentiated state of this cell was maintained.
１．試料
１）Ｃ３Ｈ１０Ｔ１／２細胞
　Ｃ３Ｈ１０Ｔ１／２細胞（理化学研究所・細胞開発銀行より入手）を、１０％　ｆｅｔａｌ　ｂｏｖｉｎｅ　ｓｅｒｕｍ，１００Ｕ／ｍｌ　ｐｅｎｉｃｉｌｌｉｎ，１００μｇ／ｍｌ　ｓｔｒｅｐｔｏｍｙｃｉｎを含む細胞培養液中（α－ＭＥＭ）で、ＣＯ２インキュベーターを用い、３７℃，５％ＣＯ２の条件下で培養した。
２）ＣＤ２７１発現ベクター
　配列表配列番号１に記載のＣＤ２７１をコードする遺伝子が配列表配列番号２に記載の発現ベクターｐＭＥ１８ＳＦＬ３に組み込まれたＴＯＹＯＢＯ　ｃＤＮＡ　Ｃｌｏｎｅｓ　ＮＧＦＲ，Ｃｏｄｅ　Ｎｏ．ＦＣＣ１１７Ｃ０２（図１）を用いた。
　図１のｃＤＮＡ部分はＣＤ２７１をコードする遺伝子（配列表配列番号１）を指す。 1. Sample 1) C3H10T1 / 2 cells C3H10T1 / 2 cells (obtained from RIKEN, Cell Development Bank) in a cell culture medium containing 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin (α-MEM) Then, using a CO 2 incubator, the cells were cultured under conditions of 37 ° C. and 5% CO 2 .
2) CD271 expression vector TOYOBO cDNA Clones NGFR, Code No. 2 in which the gene encoding CD271 described in SEQ ID NO: 1 in the sequence listing was incorporated into the expression vector pME18SFL3 described in SEQ ID NO: 2 in the sequence listing. FCC117C02 (FIG. 1) was used.
The cDNA portion in FIG. 1 refers to the gene encoding CD271 (SEQ ID NO: 1 in the sequence listing).
２．Ｃ３Ｈ１０Ｔ１／２細胞へのＣＤ２７１発現ベクターの導入
　Ｃ３Ｈ１０Ｔ１／２細胞を、１０％ｆｅｔａｌ　ｂｏｖｉｎｅ　ｓｅｒｕｍ，１００Ｕ／ｍｌ　ｐｅｎｉｃｉｌｌｉｎ，１００μｇ／ｍｌ　ｓｔｒｅｐｔｏｍｙｃｉｎを含む細胞培養液中（α－ＭＥＭ）で、５０％コンフルエント状態になるまで６ウェルディッシュで前培養した。その後、培養液を、５％ｆｅｔａｌ　ｂｏｖｉｎｅ　ｓｅｒｕｍを含む細胞培養液（α－ＭＥＭ）に交換し、２～３時間の前培養を行った。
　ＴＯＹＯＢＯ社から購入したＣ２７１発現ベクター（ＴＯＹＯＢＯ　ｃＤＮＡ　Ｃｌｏｎｅｓ　ＮＧＦＲ，Ｃｏｄｅ　Ｎｏ．ＦＣＣ１１７Ｃ０２）をＤＮＡ制限酵素ＳｃａＩで切断し、線状化ベクターを生成した。また、ｐｃＤＮＡ３．１ベクターに含まれるジェネティシン耐性遺伝子発現領域をＤＮＡ制限酵素ＫｐｎＩおよびＡｐＬＩで切り出した。
　線状化したＣＤ２７１発現ベクター（２μｇ）とｐｃＤＮＡ３．１ベクターから切り出したジェネティシン耐性遺伝子発現領域を含むＤＮＡ（０．３μｇ）をＩｎｖｉｔｒｏｇｅｎ社のｌｉｐｏｆｅｃｔａｍｉｎｅ　ＬＴＸを用いて付属のマニュアルに従って細胞にトランスフェクションした。トランスフェクションから６時間後に細胞培養液を１０％ｆｅｔａｌ　ｂｏｖｉｎｅ　ｓｅｒｕｍ，１００Ｕ／ｍｌ　ｐｅｎｉｃｉｌｌｉｎ，１００μｇ／ｍｌ　ｓｔｒｅｐｔｏｍｙｃｉｎを含む細胞培養液に交換し、２４時間培養した。その後、ジェネティシン存在下で１０日間培養した。その後、蛍光ラベルしたＣＤ２７１抗体を用いてＦＡＣＳ解析を行い、ＣＤ２７１を発現している細胞を選別した。 2. Introduction of CD271 expression vector into C3H10T1 / 2 cells C3H10T1 / 2 cells were made 50% confluent in cell culture medium containing 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin (α-MEM). Pre-cultured in 6 well dishes until complete. Thereafter, the culture solution was replaced with a cell culture solution (α-MEM) containing 5% fetal bovine serum, and pre-culture was performed for 2 to 3 hours.
A C271 expression vector (TOYOBO cDNA Clones NGFR, Code No. FCC117C02) purchased from TOYOBO was cleaved with DNA restriction enzyme ScaI to generate a linearized vector. Further, the geneticin resistance gene expression region contained in the pcDNA3.1 vector was excised with DNA restriction enzymes KpnI and ApLI.
A linearized CD271 expression vector (2 μg) and a DNA (0.3 μg) containing a geneticin resistance gene expression region excised from the pcDNA3.1 vector were transfected into cells using lipofectamine LTX of Invitrogen according to the attached manual. Six hours after transfection, the cell culture medium was replaced with a cell culture medium containing 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and cultured for 24 hours. Thereafter, the cells were cultured for 10 days in the presence of geneticin. Thereafter, FACS analysis was performed using a fluorescently labeled CD271 antibody, and cells expressing CD271 were selected.
３．Ｃ３Ｈ１０Ｔ１／２細胞（ＣＤ２７１強制発現細胞）
　導入後のＣ３Ｈ１０Ｔ１／２細胞におけるＣＤ２７１タンパク発現の確認を、試験例１に記載の方法と同様の方法で確認した。ＣＤ２７１強制発現細胞は、顕微鏡下の観察において、ＣＤ２７１発現ベクター導入による形態的な変化が認められなかった。 3. C3H10T1 / 2 cells (CD271 forced expression cells)
Confirmation of CD271 protein expression in C3H10T1 / 2 cells after introduction was confirmed by the same method as described in Test Example 1. In the CD271 forcibly expressing cells, morphological changes due to introduction of the CD271 expression vector were not observed in the observation under the microscope.
＜ＣＤ２７１強制発現細胞における未分化状態の維持の確認＞
　実施例１のＣ３Ｈ１０Ｔ１／２細胞（ＣＤ２７１強制発現細胞）を用い、１）骨芽細胞および２）脂肪細胞への分化を誘導することで、ＣＤ２７１強制発現細胞における未分化状態の維持を確認した。 <Confirmation of maintenance of undifferentiated state in cells forcibly expressing CD271>
Using the C3H10T1 / 2 cells (CD271 forced expression cells) of Example 1, the differentiation into 1) osteoblasts and 2) adipocytes was induced to confirm the maintenance of the undifferentiated state in the CD271 forced expression cells.
１）骨芽細胞への分化誘導
　実施例１のＣ３Ｈ１０Ｔ１／２細胞（ＣＤ２７１強制発現細胞）をコンフルエント状態になるまで、１０％ＦＢＳ、１００Ｕ／ｍｌ　ｐｅｎｉｃｉｌｌｉｎ、１００μｇ／ｍｌ　ｓｔｒｅｐｔｏｍｙｃｉｎを含むα－ＭＥＭ培地で３週間程度培養した。次に骨芽細胞分化誘導培地（１００ｎｇ／ｍｌ　ＢＭＰ－２、１０‐８Ｍ　Ｄｅｘａｍｅｔｈａｓｏｎｅ、５０μｇ／ｍｌ　Ｌ－ａｓｃｏｒｂａｔｅ　ｐｈｏｓｐｈａｔｅ、１０ｍＭ　β－ｇｌｙｃｅｒｏｐｈｏｓｐｈａｔｅ、１０％　ＦＢＳ、１００Ｕ／ｍｌ　ｐｅｎｉｃｉｌｌｉｎ、１００μｇ／ｍｌ　ｓｔｒｅｐｔｏｍｙｃｉｎを含むα－ＭＥＭ培地）でさらに１２日間培養し、上記試験例２と同様にアルカリフォスファターゼ染色を行った。比較としてＣＤ２７１を含まない発現ベクターのみを導入した細胞（コントロール細胞）を用いた。
　その結果、Ｃ３Ｈ１０Ｔ１／２細胞（ＣＤ２７１強制発現細胞）では、コントロール細胞と比較して顕著にアルカリフォスファターゼ活性が抑制され、骨芽細胞への分化が抑制されていることが確認された（図４）。 1) Induction of differentiation into osteoblasts In the α-MEM medium containing 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, until the C3H10T1 / 2 cells of Example 1 (CD271 forced expression cells) became confluent. Cultured for about 3 weeks. Next, osteoblast differentiation medium (100 ng / ml BMP-2, 10-8M Dexamethasone, 50 μg / ml L-ascorbate phosphate, 10 mM β-glycophosphate, 10% FBS, 100 U / ml penicillin, α containing 100 μg / ml streptomycin) -MEM medium) was further cultured for 12 days, and alkaline phosphatase staining was performed in the same manner as in Test Example 2 above. For comparison, cells into which only an expression vector not containing CD271 was introduced (control cells) were used.
As a result, in C3H10T1 / 2 cells (CD271 forced expression cells), it was confirmed that alkaline phosphatase activity was remarkably suppressed and differentiation into osteoblasts was suppressed as compared to control cells (FIG. 4). .
２）脂肪細胞への分化誘導実施例１のＣ３Ｈ１０Ｔ１／２細胞（ＣＤ２７１強制発現細胞）を上記１）と同様にコンフルエント状態になるまで培養し、その後、脂肪細胞分化誘導培地（１００ｎｇ／ｍｌ　ＢＭＰ－２、１０‐８Ｍ　Ｄｅｘａｍｅｔｈａｓｏｎｅ、１０％　ＦＢＳ、１００Ｕ／ｍｌ　ｐｅｎｉｃｉｌｌｉｎ、１００μｇ／ｍｌ　ｓｔｒｅｐｔｏｍｙｃｉｎを含むα－ＭＥＭ培地）でさらに１２日間培養し、脂肪細胞の指標とされる脂肪球の形成をＯｉｌ　ｒｅｄ　Ｏ染色によって観察した。細胞核はヘマトキシリンによって染色した。比較としてＣＤ２７１を含まない発現ベクターのみを導入した細胞（コントロール細胞）を用いた。
　その結果、Ｃ３Ｈ１０Ｔ１／２細胞（ＣＤ２７１強制発現細胞）では、コントロール細胞と比較して顕著に脂肪球の形成が抑制され、脂肪細胞への分化が抑制されていることが確認された（図５）。 2) Induction of differentiation into adipocytes The C3H10T1 / 2 cells (CD271 forced expression cells) of Example 1 were cultured until confluent as in 1) above, and then adipocyte differentiation induction medium (100 ng / ml BMP- 2, 10-8 M Dexamethasone, α-MEM medium containing 100% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin) for 12 days, and formation of fat globules as an indicator of fat cells Observed by staining. Cell nuclei were stained with hematoxylin. For comparison, cells into which only an expression vector not containing CD271 was introduced (control cells) were used.
As a result, in C3H10T1 / 2 cells (CD271 forced expression cells), it was confirmed that formation of fat globules was remarkably suppressed and differentiation into fat cells was suppressed as compared to control cells (FIG. 5). .
　上記１）、２）の結果より、Ｃ３Ｈ１０Ｔ１／２細胞（ＣＤ２７１強制発現細胞）において未分化の状態が維持されていることが示された。従って、本発明のＣＤ２７１を用いる方法により、間葉系幹細胞、ＥＳ細胞、ｉＰＳ細胞の未分化状態が維持できることが確認された。 From the results of 1) and 2) above, it was shown that an undifferentiated state was maintained in C3H10T1 / 2 cells (CD271 forced expression cells). Therefore, it was confirmed that the undifferentiated state of mesenchymal stem cells, ES cells, and iPS cells can be maintained by the method using CD271 of the present invention.
　本発明の方法を用いることにより、未分化状態が長期的に維持された間葉系幹細胞、ＥＳ細胞、ｉＰＳ細胞を提供することができる。また、ＣＤ２７１を発現するベクターや、細胞膜透過化型リコンビナントＣＤ２７１タンパク質を、これらの細胞の未分化状態維持物質として提供することもできる。さらに、ＣＤ２７１を有効成分とする細胞分化の抑制および未分化状態を維持するための組成物を用い、医薬品（例えば脂肪細胞分化を抑制することによるメタボリックシンドローム治療のための医薬品等）を提供することもできる。 By using the method of the present invention, it is possible to provide mesenchymal stem cells, ES cells, and iPS cells whose undifferentiated state has been maintained for a long time. In addition, a vector that expresses CD271 and a cell membrane permeabilizing recombinant CD271 protein can also be provided as substances that maintain the undifferentiated state of these cells. Furthermore, using a composition for suppressing cell differentiation and maintaining an undifferentiated state containing CD271 as an active ingredient, to provide a pharmaceutical (eg, a pharmaceutical for treating metabolic syndrome by inhibiting adipocyte differentiation). You can also.

Claims (9)

  1. 未分化状態の細胞にＣＤ２７１発現ベクターまたは細胞膜透過化型リコンビナントＣＤ２７１タンパク質を導入することによって、細胞の未分化状態を維持する方法。 A method of maintaining an undifferentiated state of a cell by introducing a CD271 expression vector or a cell membrane permeabilizing recombinant CD271 protein into an undifferentiated cell.
  2. ＣＤ２７１発現ベクターが配列表配列番号１に記載のＣＤ２７１をコードする遺伝子の全部または一部が組み込まれた発現ベクターである請求項１に記載の方法。 The method according to claim 1, wherein the CD271 expression vector is an expression vector into which all or part of the gene encoding CD271 described in SEQ ID NO: 1 of the Sequence Listing is incorporated.
  3. ＣＤ２７１発現ベクターが図１に記載のＣＤ２７１発現ベクターである請求項１または２に記載の方法。 The method according to claim 1 or 2, wherein the CD271 expression vector is the CD271 expression vector shown in FIG.
  4. 未分化状態の細胞が、間葉系幹細胞、ＥＳ細胞またはｉＰＳ細胞のいずれかである請求項１～３のいずれかに記載の方法。 The method according to any one of claims 1 to 3, wherein the undifferentiated cell is a mesenchymal stem cell, an ES cell or an iPS cell.
  5. 請求項１～４のいずれかに記載の方法によって、未分化状態が維持された細胞。 A cell in which an undifferentiated state is maintained by the method according to any one of claims 1 to 4.
  6. 細胞の未分化状態を維持するためのＣＤ２７１発現ベクターまたは細胞膜透過化型リコンビナントＣＤ２７１タンパク質。 A CD271 expression vector or a cell membrane permeabilizing recombinant CD271 protein for maintaining an undifferentiated state of a cell.
  7. ＣＤ２７１発現ベクターまたは細胞膜透過化型リコンビナントＣＤ２７１タンパク質のいずれか１つ以上を含む細胞の未分化状態を維持するためのキット。 A kit for maintaining an undifferentiated state of a cell comprising any one or more of a CD271 expression vector or a cell membrane permeabilized recombinant CD271 protein.
  8. ＣＤ２７１を有効成分とする、細胞分化を抑制する、または細胞の未分化状態を維持するための組成物。 A composition containing CD271 as an active ingredient, for inhibiting cell differentiation or maintaining an undifferentiated state of cells.
  9. 請求項８に記載の組成物を含む、脂肪細胞への細胞分化を抑制することによるメタボリックシンドローム対処用の医薬品。 A pharmaceutical product for treating metabolic syndrome by inhibiting cell differentiation into adipocytes, comprising the composition according to claim 8.
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