WO2016017795A1 - Method for evaluating quality of human mesenchymal stem cell, and monoclonal antibody for use in said method - Google Patents

Method for evaluating quality of human mesenchymal stem cell, and monoclonal antibody for use in said method Download PDF

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WO2016017795A1
WO2016017795A1 PCT/JP2015/071770 JP2015071770W WO2016017795A1 WO 2016017795 A1 WO2016017795 A1 WO 2016017795A1 JP 2015071770 W JP2015071770 W JP 2015071770W WO 2016017795 A1 WO2016017795 A1 WO 2016017795A1
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mesenchymal stem
human mesenchymal
stem cells
cells
cell
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PCT/JP2015/071770
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French (fr)
Japanese (ja)
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有未 伊谷
岡野 栄之
洋 馬渕
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有未 伊谷
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Priority to CA2954245A priority Critical patent/CA2954245C/en
Priority to SG11201610795VA priority patent/SG11201610795VA/en
Priority to AU2015297347A priority patent/AU2015297347B2/en
Priority to ES15827606T priority patent/ES2927112T3/en
Priority to EP15827606.3A priority patent/EP3176253B1/en
Priority to US15/321,679 priority patent/US20170204374A1/en
Priority to JP2016538462A priority patent/JP6850944B2/en
Publication of WO2016017795A1 publication Critical patent/WO2016017795A1/en
Priority to US17/190,192 priority patent/US11441123B2/en

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    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0652Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662Stem cells
    • C12N5/0663Bone marrow mesenchymal stem cells (BM-MSC)
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
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    • C12Y207/00Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10Protein-tyrosine kinases (2.7.10)
    • C12Y207/10001Receptor protein-tyrosine kinase (2.7.10.1)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966Animal cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705Assays involving receptors, cell surface antigens or cell surface determinants
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/91Transferases (2.)
    • G01N2333/912Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • the present invention relates to a human mesenchymal stem cell quality evaluation method, a human mesenchymal stem cell isolation, selection and culture method, a rapidly proliferating human mesenchymal stem cell population, and a rapidly proliferating human mesenchymal stem cell. It relates to a monoclonal antibody that specifically recognizes.
  • MSC Mesenchymal stem cells
  • Non-Patent Documents 2 and 3, and Patent Document 1 using antibodies against CD271 (LNGFR) and CD90 (Thy1), LNGFR Thy1 co-positive from human bone marrow, placental chorion, adipose tissue, peripheral blood, dental pulp, etc. By selecting cells, human mesenchymal stem cells can be concentrated.
  • LNGFR CD271
  • Thy1 CD90
  • the selected LNGFR Thy1 co-positive cells are cultured in a single cell (clone), and a rapidly growing lot (REC: Rapidly Expanding Clone) is selected.
  • Leaf stem cells can be obtained.
  • High-purity human mesenchymal stem cells (REC) obtained by the same method had 1000 times or more the ability of proliferation, differentiation and migration as compared with mesenchymal stem cells obtained by the conventional method.
  • REC Rapidly Expanding Clone
  • human mesenchymal stem cell quality evaluation method aims at providing the monoclonal antibody which recognizes specifically.
  • the quality evaluation method for human mesenchymal stem cells is characterized by isolating, selecting and culturing rapidly proliferating human mesenchymal stem cells from a cell population containing human mesenchymal stem cells. Then, the cell population expressing Ror2 or Fzd5 in the separated, sorted, and cultured cell population is quantified (presence ratio), and pass / fail judgment of each cell population is performed. According to this configuration, Ror2 or Fzd5 can determine whether or not it is REC by single expression, and since cultured cells can be used, determination can be performed more easily. It should be noted that LNGFR is not expressed in cultured cells even if it is REC, and Thy1 alone cannot determine REC.
  • cells expressing Ror2 or Fzd5 may be quantified using an anti-Ror2 monoclonal antibody or an anti-Fzd5 monoclonal antibody.
  • Ror2 mRNA expression may be quantified using quantitative PCR, or cells expressing Ror2 or Fzd5 may be quantified by immunostaining.
  • FMC can be easily applied.
  • Fzd5 is expressed intracellularly, visual evaluation of immunostaining is appropriate.
  • human mesenchymal stem cells are isolated, sorted and cultured by separating, selecting and selecting rapidly proliferating human mesenchymal stem cells from a cell population containing human mesenchymal stem cells.
  • To determine the pass / fail of each cell population by quantifying (the abundance ratio) of Ror2 or Fzd5 expressing cells in the cultured, separated, sorted and cultured cell population, and to select only the pass cell population. is there.
  • cells expressing Ror2 or Fzd5 may be quantified using an anti-Ror2 monoclonal antibody or an anti-Fzd5 monoclonal antibody.
  • the cells expressing Ror2 mRNA may be quantified using quantitative PCR, or the cells expressing Ror2 or Fzd5 may be quantified by immunostaining.
  • the step of separating, selecting and culturing as the rapidly proliferating human mesenchymal stem cells is performed by performing flow cytometry on cells stained with anti-LNGFR monoclonal antibody and anti-Thy1 monoclonal antibody from a cell population containing human mesenchymal stem cells.
  • FCM flow cytometry on cells stained with anti-LNGFR monoclonal antibody and anti-Thy1 monoclonal antibody from a cell population containing human mesenchymal stem cells.
  • the step of separating, selecting and culturing the human mesenchymal stem cells as rapidly proliferating comprises analyzing cells stained with an anti-Ror2 monoclonal antibody by FCM from a cell population containing human mesenchymal stem cells, A step of cell sorting Ror2-positive cells may be included.
  • each of the above methods may include a step of directly preparing the cell population from cells derived from tissues such as bone marrow.
  • the method including the step of cell sorting Ror2-positive cells may include the step of preparing the cell population by attaching and culturing tissue-derived cells such as bone marrow. This is because cultured cells of REC do not become LNGFR positive but become Ror2 positive.
  • the cell sorting step may be a step of seeding each positive cell in a well of a culture plate, and further including a step of separating and selecting cells in a well that has become confluent by culture. Good.
  • the characteristics of a cell population of fast-growing human mesenchymal stem cells are characterized by separating, selecting and culturing fast-growing human mesenchymal stem cells from a cell population containing human mesenchymal stem cells, In the separated, sorted, and cultured cell population, the cells expressing Ror2 or Fzd5 (presence ratio thereof) are quantified, and pass / fail judgment of each cell population is performed, and only the passed cell population is selected.
  • cells stained with an anti-LNGFR monoclonal antibody and an anti-Thy1 monoclonal antibody from a cell population containing human mesenchymal stem cells are analyzed by FCM, and LNGFR Thy1 co-positive cells are cell-sorted into the wells of a culture plate.
  • the quantification may be performed after seeding the cells that are co-positive and separating and sorting the cells in the wells that have become confluent by culture.
  • the novel monoclonal antibody according to the present invention is an anti-Ror2 monoclonal antibody, and the clone name is 7C9.
  • Another novel monoclonal antibody according to the present invention is an anti-Fzd5 monoclonal antibody, and the clone name is 6F5.
  • the expression of the protein encoded by the two specified genes is specific to REC and is not observed in the cell population with degraded quality. It is an essential gene for maintaining the undifferentiated state of REC. 1) Cell performance degradation is induced by inhibition of expression. 2) Undifferentiated state is prolonged by forced expression of gene. Rather, it is closely related to cell function and is an effective index that guarantees cell performance.
  • human mesenchymal stem cell quality evaluation method human mesenchymal stem cell isolation, selection and culture method, rapidly proliferating human mesenchymal stem cell population, and rapid proliferation It has become possible to provide monoclonal antibodies that specifically recognize human mesenchymal stem cells.
  • FIG. 2 is a graph and graph showing that inhibition of Fzd5 induces degradation of the cellular properties of REC. It is the photograph and graph which show that forced expression of Fzd5 induces the undifferentiation of REC. It is the photograph and graph which show the dyeing
  • FIG. 1 illustrates a REC separation step by a monoclonal culture method.
  • Mononuclear cells are prepared from human bone marrow (or fat / placental chorion), and bone marrow mononuclear cells are stained with anti-LNGFR and anti-Thy1 (LNGFR Thy1 co-positive cells are referred to as LT cells).
  • FCM flow cytometry
  • an anti-CD106 monoclonal antibody may be added, and LNGFR positive / Thy1 positive and CD106 strong positive cells may be clonal-sorted.
  • REC Rapidly Expanding Cells
  • MEC / SEC Modely / Slowly Expanding Cells
  • FIG. 1 further shows evaluation of cultured cells using a REC marker (anti-Ror2 / anti-Fzd5).
  • REC recovered from the 96-well plate is transferred to each culture dish or culture flask for each well and cultured until confluent (expansion culture).
  • cells that adhere and proliferate from all lots are collected, and a part of each lot (about 1 to 3 ⁇ 10 3 cells), especially monoclonal antibodies against REC markers (anti-Ror2 and anti-Fzd5) Perform single staining with.
  • REC marker positive cells by flow cytometry, and determining the ratio of REC marker positive cells in the recovered cells (Ror2 mRNA expression may be quantified using quantitative PCR, or the ratio may be manually determined using a microscope. May be determined by: 4) A lot (cell population) in which the positive ratio is a certain value (for example, 65%) or more is accepted. 5) Enclose the acceptable lot of cells in a frozen vial and store in liquid nitrogen. 6) The frozen cells are used as high-quality human mesenchymal stem cells (product). 7) After thawing the cells in the vial, the user can cultivate the cells on a culture dish or flask and finally use a high-purity mesenchymal stem cell of 1 ⁇ 10 10 or more stably. It is.
  • Ror2-positive cells may be clone-sorted instead of clone-sorting LT cells (Table 1, P6, 7).
  • LT cells and Ror2-positive cells may be selected and seeded in each well of a 96-well culture plate (indicated as “plurality” in Table 1, P5, 9, 14). Purity is lower than sorting.
  • the confluent is a state in which the cultured cells cover 90% or more of the surface of the culture container.
  • Semi-confinent is a state in which cultured cells cover 70-80% of the surface of the culture vessel. The size and type of the culture instrument to be used can be appropriately changed according to the cell growth rate.
  • the cell population is prepared directly from cells derived from tissues such as bone marrow.
  • the cells are obtained by attaching and culturing cells derived from tissues such as bone marrow.
  • Populations may be prepared (Table 1, P10-15, labeled “adherent cultured cells”).
  • bone marrow mononuclear cells are seeded on a medium containing 10-20% serum + bFGF (37 ° C., 1-5% CO 2 ) and cultured for about 2 weeks, and fibroblast-like adherent cells appearing after the culture.
  • the step of preparing the cell population may include a step of treating the bone marrow with collagenase.
  • a cell population may be prepared from peripheral blood after administration of G-CSF.
  • FIG. A shows the results of FCM analysis after staining human bone marrow mononuclear cells with antibodies against LNGFR and Thy1. The portion surrounded by the ellipse is the LT cell.
  • FIG. B is a schematic diagram of single cell separation (clone sorting) of LT cells in a 96-well plate.
  • FIG. C is a graph showing the results of periodically measuring the number of cells after single cell culture. REC has a faster growth rate than MEC / SEC and grows to 0.5-1 ⁇ 10 4 cells in about 2 weeks.
  • FIG. D shows the results of quantitative expression PCR measuring gene expression specific to bone and fat cells after inducing differentiation of REC, MEC and SEC into bone and fat. REC was shown to have particularly high fat differentiation ability compared to MEC and SEC.
  • FIG. E is a graph showing the results of comparing the number of wells in which secondary colonies were formed after REC / MEC / SEC were again cloned into a 96-well plate. The formation of secondary colonies is an index of self-replication ability, which is a measure of the undifferentiated state. About 33% of RECs form secondary colonies, whereas MEC / SEC forms very few colonies.
  • FIG. D shows the results of quantitative expression PCR measuring gene expression specific to bone and fat cells after inducing differentiation of REC, MEC and SEC into bone and fat. REC was shown to have particularly high fat differentiation ability compared to MEC and SEC.
  • FIG. E is a graph showing the results of comparing the number of wells in which secondary colonies were formed after REC
  • F shows the following cell population introduced with a Luc (luciferase) gene expression vector, WBM (MSC obtained by a conventional method, WBM is an abbreviation for Whole Bone Marrow), REC, MEC / SEC, and negative control group
  • WBM Luc (-) Cultured MSC
  • IVIS device
  • the upper graph is a graph in which the amount of Luc luminescence in each mouse is digitized, and the luminescence ratio (%) of mice transplanted with other cells when the WBM / MSC transplanted group is taken as 100% is plotted.
  • the image is an image of Luc luminescence in each group of recipient mice. From both results, REC-transplanted mice have extremely low light emission in the lung, so REC is hardly trapped in lung capillaries, whereas MEC / SEC is WBM (cultured MSC obtained above). ) Is supplemented almost equivalently, and remains in the lungs.
  • REC is a cell population with excellent proliferation ability, differentiation ability, and migration ability, and especially migratory ability comparable to MSC in fresh bone marrow in that it can be systemically administered to intractable diseases described later. The point is to maintain.
  • REC has the following characteristics compared to normal MSC. 1.
  • Morphologically extremely uniform cell population 2.
  • Amplification of culture is possible with high division rate and maintaining undifferentiation.
  • Cell population with high differentiation potential and easy to differentiate into bone and fat REC, which maintains its migration ability is the most undifferentiated cell population among human MSCs, and has properties closest to those in bone marrow.
  • MSC obtained by MEC / SEC or conventional methods, it is a cell population with excellent cell differentiation, proliferation, and migration ability, freshness, low mutation, and guaranteed cell performance.
  • FIG. A is a comparison of Ror2 mRNA expression by quantitative PCR in each of REC, MEC, and SEC.
  • FIG. B is a comparison of Fzd5 mRNA expression by quantitative PCR in each of REC, MEC, and SEC.
  • FIG. C is a comparison of Fzd5 protein expression by Western blotting.
  • D is a photograph comparing the intracellular localization of Fzd5 protein by fluorescent immunostaining.
  • Fzd5 and Ror2 is REC-specific. Therefore, if the expression of each of Fzd5 and Ror2 is detected and quantified, it is considered effective as an index for evaluating the cell quality of REC.
  • the newly produced anti-Fzd5 monoclonal antibody and anti-Ror2 monoclonal antibody can detect and quantify the target protein antigen in any of flow cytometry, western blotting, and fluorescent immunostaining.
  • the RNA interlacing method is a method for examining the function of a target gene by introducing a short RNA (shRNA) having a sequence complementary to the target mRNA into the cell and destroying the target mRNA.
  • shRNA short RNA
  • results of a series of experiments comparing the cellular properties of REC when Fzd5 mRNA was disrupted by shRNA having a sequence complementary to Fzd5 (shFZD5) with the control group (shCTRL: shRNA having a random sequence not complementary to Fzd5) Indicates. Fig.
  • FIG. A is a graph in which the amount of Fzd5 mRNA was quantified by quantitative PCR after introducing shFZD5 or shCTRL into REC.
  • the amount of Fzd5 mRNA in the control group shCTRL, short hairpin control
  • the amount of Fzd5 mRNA was reduced to about 40% in the REC in which shFZD5 was forcibly expressed.
  • Fig. B is a graph in which, after introducing shFZD5 or shCTRL into REC, the number of cells in the shFZD5 forced expression group when the number of cells in the control group is 1 is plotted on the vertical axis, and the number of days after shRNA introduction is plotted on the horizontal axis. It is.
  • Fig. C is an image of shFZD5 or shCTRL introduced into REC, induced to differentiate into adipocytes, and stained with Oil-Red-O on the 14th day of culture.
  • Fzd5 was inhibited as compared with the control group, a decrease in fat differentiation ability was observed.
  • SA- ⁇ -gal activity which is an indicator of cell senescence, can be detected by staining with blue when the substrate X-gal is added.
  • FIG. E is a graph in which the amount of mRNA of p16 (INK4a), which is an indicator of cell senescence, is quantified by a quantitative PCR method.
  • the control group was 100
  • the amount of p16 mRNA in shFZD5-introduced REC was about 300, indicating that cell senescence was induced by inhibition of Fzd5 expression.
  • F shows images obtained by introducing shFZD5 or shCTRL into each REC and then staining each cell population with intracellular anti-F-actin antibody to observe the formation of Stress Fiber, and each of the cell populations.
  • the graph which plotted the average value of the area (cell size) of a cell is shown. From the above results, inhibition of the function of Fzd5 in REC leads to a decrease in proliferation ability, a decrease in differentiation ability, induction of cell senescence, a decrease in migration due to stress fiber formation and an increase in cell size, and MEC / Since it changes to the same property as SEC, Fzd5 is not a mere biomarker, but is considered to be a functional molecule that ensures the maintenance of the cell performance of REC.
  • FIG. 5A is a morphological observation photograph of GFP-expressing cells using a fluorescence microscope.
  • FIG. B is a graph in which the number of Fzd5-expressing REC cells is plotted on the vertical axis and the number of days after gene introduction is plotted on the horizontal axis when the number of cells in the control group is 1.
  • FIG. B shows the results of Western blotting in which intracellular proteins were prepared from 3 clones with different RECs, and Fzd5 protein was detected using 6F5 as a primary antibody.
  • 6F5 an intracellular protein prepared from monkey kidney-derived cell line COS7 was used.
  • FIG. C is an image of REC cells stained with 6F5-Biotin as a primary antibody, observed with a fluorescent microscope after streptavidin (SAV) -Alexa555 and labeled with a fluorescent microscope.
  • Anti-Fzd5 antibody (6F5) was available for all of flow cytometry, Western blotting, and fluorescent immunostaining.
  • FIG. A novel anti-Ror2 antibody (clone name: 7C9) was prepared using human Ror2 antigen as an immunogen.
  • Fig. A shows the results of staining REC with 7C9-Biotin as a primary antibody, fluorescently labeling with SAV-PE, and detecting PE fluorescence using flow cytometry.
  • a sample to which an isotype control antibody is added as a primary antibody is used as a negative control. It is a two-dimensional dot plot in which the vertical axis is FITC fluorescence (all negative because it is not stained), and the horizontal axis is PE fluorescence.
  • FIG. B is an image obtained by performing immunostaining on REC using 7C9-Biotin as a primary antibody, followed by fluorescence labeling with Streptavidin-Alexa488, and observing and photographing the expression of Ror2 protein with a fluorescence microscope. It was confirmed that most of REC expressed Ror2 protein.
  • 3C shows the results of flow cytometry analysis of fresh bone marrow cells by triple staining with LNGFR-APC, Thy1-FITC, Ror2-PE (monoclonal antibodies against each antigen).
  • LNGFR-APC Lignin-APC
  • Thy1-FITC Thy1-FITC
  • Ror2-PE monoclonal antibodies against each antigen.
  • LNGFR expression is plotted on the vertical axis
  • Thy1 expression is plotted on the horizontal axis
  • the portion surrounded by a square frame is a LNGFR Thy1 co-positive cell population containing human MSCs at a high frequency.
  • FIG. 1 The two figures on the right show only the LNGFR Thy1 co-positive cell population, and the horizontal axis shows the FSC, which is an indicator of cell size, and the vertical axis shows the PE fluorescence of the anti-Ror2-Biotin antibody (7C9) labeled with SAV-PE.
  • FIG. The portion surrounded by a square frame is a Ror2 positive region set based on the negative control, and the numerical value represents the positive rate (%).
  • Clone7C9 92.3% of LNGFR Thy1 co-positive cells are Ror2 positive and can be used as a selection marker for MCS in place of LNGFR Thy1.
  • Ror2 protein By using a newly prepared anti-Ror2 monoclonal antibody, Ror2 protein can be detected and quantified by flow cytometry and fluorescent immunostaining (FIGS. 7A and 7B). Furthermore, the newly produced anti-Ror2 can be used as a marker for MSC contained in the bone marrow (FIG. 7C).
  • the frequency (% content) of positive cells is measured by flow cytometry. Instead, the frequency (content%) of positive cells may be measured under a fluorescence microscope.
  • the Ror2 positive rate of REC was 72% ⁇ 8.9% in 5 lots. Therefore, for example, the minimum value of 63% or more or 65% or more may be used as the reference value for the pass / fail determination.
  • the present invention it is possible to provide a technique for efficiently separating and culturing human mesenchymal stem cells that can be used for the treatment of systemic diseases, and whether the obtained cell population is suitable for transplantation, or a medicinal effect. It is now possible to perform quality evaluations that serve as the basis for indicating Among newly-prepared monoclonal antibodies that exhibit staining properties specialized for high-purity mesenchymal stem cells, candidates suitable for cell separation can be commercialized as reagents for separating mesenchymal stem cells by binding to nanomagnetic particles. . In addition, fluorescent substance-bound antibodies and cell staining reagents can be put to practical use as cell evaluation reagents for examining the quality of the separated mesenchymal stem cells.
  • Mesenchymal stem cells are used for biomaterials that have been used in the past, or by taking advantage of their multipotency, administration to myasthenia gravis, chronic rheumatism, etc., spinal cord injury, cardiovascular, chronic liver failure
  • Various applications are expected, such as co-transplantation as supporting cells to prepare the tissue (niche) when performing cell therapy for severe disease treatment such as.
  • REC that maintains migratory properties
  • metabolic diseases such as systemic bone and cartilage diseases such as hypophosphatasia, for which treatment has not been available
  • intravenous treatment such as treatment of GVHD If it is applied to all diseases that need to be administered, the therapeutic effect is unprecedented.

Abstract

The purpose of the present invention is to provide: a method for evaluating the quality of a human mesenchymal stem cell; a method for separating, selecting and culturing a human mesenchymal stem cell; a human mesenchymal stem cell population which can proliferate rapidly; and a monoclonal antibody which can specifically recognize a human mesenchymal stem cell that proliferates rapidly. Human mesenchymal stem cells that proliferate rapidly are separated and selected from a cell population containing human mesenchymal stem cells, and are then cultured. The abundance ratio of cells each having Ror2 or Fzd5 expressed therein in the cell population that has been separated, selected and cultured is quantified to determine whether or not the cell population passes or fails.

Description

[規則37.2に基づきISAが決定した発明の名称] ヒト間葉系幹細胞の品質評価方法、及び、そのためのモノクローナル抗体[Name of invention determined by ISA based on Rule 37.2] Quality evaluation method of human mesenchymal stem cells and monoclonal antibody therefor
 本発明は、ヒト間葉系幹細胞の品質評価方法、ヒト間葉系幹細胞の分離、選別及び培養方法、増殖の早いヒト間葉系幹細胞の細胞集団、並びに、増殖の早いヒト間葉系幹細胞を特異的に認識するモノクローナル抗体に関する。 The present invention relates to a human mesenchymal stem cell quality evaluation method, a human mesenchymal stem cell isolation, selection and culture method, a rapidly proliferating human mesenchymal stem cell population, and a rapidly proliferating human mesenchymal stem cell. It relates to a monoclonal antibody that specifically recognizes.
 間葉系幹細胞 (Mesenchymal Stem Cells : MSC) は細胞採取に伴う倫理的問題が少なく、骨・軟骨・脂肪などへの多様な分化能を持つことから、造血幹細胞に次いで臨床応用が盛んに行われている体性幹細胞の一つである。後述する比較的簡単な手技により分離できることから、主に試験管内で軟骨・骨などへ分化誘導後に局所へ移植するなど、バイオマテリアルの材料として広く用いられている。
 ヒト間葉系幹細胞の分離培養方法としては、非特許文献1で報告されている培養法が一般的に用いられる。しかし従来法で得た細胞集団には劣化した(分化・増殖・遊走能を失った)夾雑細胞が多数混入しており、この夾雑物が本来はポテンシャルを持つはずの細胞に影響し、さらなる品質の劣化をまねく要因となる。
 かかる従来の実情に鑑みて、増殖能・分化能・遊走能が従来よりも優れたヒト間葉系幹細胞の分離培養方法を確立した(非特許文献2、3、特許文献1)。これらの非特許文献2、3及び特許文献1によれば、CD271(LNGFR)とCD90(Thy1)に対する抗体を用い、ヒト骨髄、胎盤絨毛膜、脂肪組織、末梢血、歯髄などよりLNGFR Thy1共陽性細胞を選別することでヒト間葉系幹細胞を濃縮することができる。
Mesenchymal stem cells (MSC) have few ethical problems associated with cell collection and have a variety of differentiating potentials such as bone, cartilage, and fat. Is one of the somatic stem cells. Since it can be separated by a relatively simple procedure, which will be described later, it is widely used as a material for biomaterials such as transplantation to a local site after induction of differentiation into cartilage, bone, etc. mainly in a test tube.
As a method for separating and culturing human mesenchymal stem cells, the culture method reported in Non-Patent Document 1 is generally used. However, the cell population obtained by the conventional method is contaminated with many contaminated cells that have deteriorated (differentiated, proliferated, or migrated), and this contaminant affects the cells that should originally have potential. It becomes a factor that leads to deterioration.
In view of the conventional situation, a method for isolating and culturing human mesenchymal stem cells having superior proliferation ability, differentiation ability, and migration ability as compared with the conventional methods has been established (Non-patent Documents 2 and 3, Patent Document 1). According to these Non-Patent Documents 2 and 3, and Patent Document 1, using antibodies against CD271 (LNGFR) and CD90 (Thy1), LNGFR Thy1 co-positive from human bone marrow, placental chorion, adipose tissue, peripheral blood, dental pulp, etc. By selecting cells, human mesenchymal stem cells can be concentrated.
 また選別したLNGFR Thy1共陽性細胞を単一細胞(クローン)培養し、増殖が速いロット(REC: Rapidly Expanding Clone)を選択することで、増殖能・分化能・遊走能にすぐれた高純度ヒト間葉系幹細胞を得ることが可能となった。
 同方法で得た高純度ヒト間葉系幹細胞(REC)は従来法で得た間葉系幹細胞と比較し、増殖能・分化能・遊走能全てが1000倍以上の能力を持っていた。
 上記ヒト間葉系幹細胞の分離培養方法の特徴によれば、単一細胞培養を行うことで夾雑細胞を含まない条件が形成され、細胞品質を維持した拡大培養が可能となる。特に遊走能を保持しているため、経静脈内投与が可能となり、骨・軟骨形成不全症等の重篤な全身性疾患への応用が期待できるようになった。
In addition, the selected LNGFR Thy1 co-positive cells are cultured in a single cell (clone), and a rapidly growing lot (REC: Rapidly Expanding Clone) is selected. Leaf stem cells can be obtained.
High-purity human mesenchymal stem cells (REC) obtained by the same method had 1000 times or more the ability of proliferation, differentiation and migration as compared with mesenchymal stem cells obtained by the conventional method.
According to the characteristics of the above-described method for separating and culturing human mesenchymal stem cells, conditions that do not include contaminating cells are formed by performing single cell culture, and expansion culture that maintains cell quality is possible. In particular, since it has the ability to migrate, it can be administered intravenously, and can be expected to be applied to serious systemic diseases such as bone and cartilage dysplasia.
特開2009-60840号公報JP 2009-60840 A
 不死化細胞株とは異なり、RECといえども継代培養を長期に繰り返すことによる細胞品質の劣化は避けられないが、現時点では品質劣化の明確な指標が存在しない。 異 な り Unlike immortalized cell lines, even RECs cannot avoid deterioration of cell quality due to repeated subculture for a long time, but at present there is no clear indicator of quality deterioration.
 本発明では、ヒト間葉系幹細胞の品質評価方法、ヒト間葉系幹細胞の分離、選別及び培養方法、増殖の早いヒト間葉系幹細胞の細胞集団、並びに、増殖の早いヒト間葉系幹細胞を特異的に認識するモノクローナル抗体を提供することを目的とする。 In the present invention, human mesenchymal stem cell quality evaluation method, human mesenchymal stem cell isolation, selection and culture method, rapidly proliferating human mesenchymal stem cell population, and rapidly proliferating human mesenchymal stem cell It aims at providing the monoclonal antibody which recognizes specifically.
 また、RECとそれ以外の劣化したクローン(MEC: Moderately Expanding Clone, SEC: Slowly Expanding Clone)とで遺伝子発現解析を行い、REC特異的遺伝子を選別した。さらに特異的遺伝子が発現する蛋白を認識する新規モノクローナル抗体の作製を目的とする。 Also, gene expression analysis was performed on REC and other degraded clones (MEC: “Moderately” Expanding “Clone”, “SEC:“ Slowly ”Expanding” Clone), and REC-specific genes were selected. Furthermore, it aims at preparation of the novel monoclonal antibody which recognizes the protein which a specific gene expresses.
 上記目的を達成するため、本発明に係るヒト間葉系幹細胞の品質評価方法の特徴は、ヒト間葉系幹細胞が含まれる細胞集団から、増殖の早いヒト間葉系幹細胞を分離、選別及び培養し、同分離、選別及び培養した細胞集団においてRor2またはFzd5を発現している細胞(の存在比率)を定量して各細胞集団の合否判定を行うことにある。同構成によれば、Ror2またはFzd5は、単独発現によりRECであるか否かを判定でき、培養細胞も利用可能であるため、判定をより簡便に行うことができる。なお、LNGFRは培養細胞ではRECであっても発現せず、Thy1は単独ではREC判定不能である。 In order to achieve the above object, the quality evaluation method for human mesenchymal stem cells according to the present invention is characterized by isolating, selecting and culturing rapidly proliferating human mesenchymal stem cells from a cell population containing human mesenchymal stem cells. Then, the cell population expressing Ror2 or Fzd5 in the separated, sorted, and cultured cell population is quantified (presence ratio), and pass / fail judgment of each cell population is performed. According to this configuration, Ror2 or Fzd5 can determine whether or not it is REC by single expression, and since cultured cells can be used, determination can be performed more easily. It should be noted that LNGFR is not expressed in cultured cells even if it is REC, and Thy1 alone cannot determine REC.
 上記構成において、抗Ror2モノクローナル抗体または抗Fzd5モノクローナル抗体を用いてRor2またはFzd5を発現している細胞を定量するとよい。この場合、定量的PCRを用いてRor2のmRNA発現を定量してもよいし、免疫染色によりRor2またはFzd5を発現している細胞を定量してもよい。ただしRor2は細胞外発現であるため、FMCの適用が容易であるが、Fzd5は細胞内発現であるため、免疫染色の目視評価等が適切である。 In the above configuration, cells expressing Ror2 or Fzd5 may be quantified using an anti-Ror2 monoclonal antibody or an anti-Fzd5 monoclonal antibody. In this case, Ror2 mRNA expression may be quantified using quantitative PCR, or cells expressing Ror2 or Fzd5 may be quantified by immunostaining. However, since Ror2 is expressed extracellularly, FMC can be easily applied. However, since Fzd5 is expressed intracellularly, visual evaluation of immunostaining is appropriate.
 一方、上記目的を達成するため、ヒト間葉系幹細胞の分離、選別及び培養方法の特徴は、ヒト間葉系幹細胞が含まれる細胞集団から、増殖の早いヒト間葉系幹細胞を分離、選別及び培養し、同分離、選別及び培養した細胞集団においてRor2またはFzd5を発現している細胞(の存在比率)を定量して各細胞集団の合否判定を行い、合格の細胞集団のみを選別することにある。 On the other hand, in order to achieve the above object, human mesenchymal stem cells are isolated, sorted and cultured by separating, selecting and selecting rapidly proliferating human mesenchymal stem cells from a cell population containing human mesenchymal stem cells. To determine the pass / fail of each cell population by quantifying (the abundance ratio) of Ror2 or Fzd5 expressing cells in the cultured, separated, sorted and cultured cell population, and to select only the pass cell population. is there.
 同構成において、抗Ror2モノクローナル抗体または抗Fzd5モノクローナル抗体を用いてRor2またはFzd5を発現している細胞を定量するとよい。ここで、定量的PCRを用いてRor2のmRNAを発現している細胞を定量してもよいし、免疫染色によりRor2またはFzd5を発現している細胞を定量してもよい。 In the same configuration, cells expressing Ror2 or Fzd5 may be quantified using an anti-Ror2 monoclonal antibody or an anti-Fzd5 monoclonal antibody. Here, the cells expressing Ror2 mRNA may be quantified using quantitative PCR, or the cells expressing Ror2 or Fzd5 may be quantified by immunostaining.
 また、前記増殖の早いヒト間葉系幹細胞として分離、選別及び培養する工程が、ヒト間葉系幹細胞が含まれる細胞集団から、抗LNGFRモノクローナル抗体および抗Thy1モノクローナル抗体で染色した細胞をフローサイトメトリー(以下、「FCM」)で解析し、LNGFR Thy1共陽性細胞をセルソーティングする工程を含んでいてもよい。これに限らず、前記増殖の早いヒト間葉系幹細胞として分離、選別及び培養する工程が、ヒト間葉系幹細胞が含まれる細胞集団から、抗Ror2モノクローナル抗体で染色した細胞をFCMで解析し、Ror2陽性細胞をセルソーティングする工程を含んでいてもよい。 In addition, the step of separating, selecting and culturing as the rapidly proliferating human mesenchymal stem cells is performed by performing flow cytometry on cells stained with anti-LNGFR monoclonal antibody and anti-Thy1 monoclonal antibody from a cell population containing human mesenchymal stem cells. (Hereinafter referred to as “FCM”), and may include a step of cell sorting LNGFR Thy1 co-positive cells. Without being limited thereto, the step of separating, selecting and culturing the human mesenchymal stem cells as rapidly proliferating comprises analyzing cells stained with an anti-Ror2 monoclonal antibody by FCM from a cell population containing human mesenchymal stem cells, A step of cell sorting Ror2-positive cells may be included.
 さらに、上記各方法において、骨髄を始めとする各組織由来細胞から直接前記細胞集団を調製する工程を包含してもよい。一方、Ror2陽性細胞をセルソーティングする工程を含む方法では、骨髄をはじめとする組織由来細胞を付着培養することにより、前記細胞集団を調製する工程を包含してもよい。RECの培養細胞はLNGFR陽性とならない一方、Ror2陽性となるからである。 Furthermore, each of the above methods may include a step of directly preparing the cell population from cells derived from tissues such as bone marrow. On the other hand, the method including the step of cell sorting Ror2-positive cells may include the step of preparing the cell population by attaching and culturing tissue-derived cells such as bone marrow. This is because cultured cells of REC do not become LNGFR positive but become Ror2 positive.
 上記方法において、前記セルソーティングする工程が、培養プレートのウェルに陽性の各細胞を播種するものであり、その後さらに、培養によりコンフルエントとなったウェルの細胞を分離及び選別する工程を包含させてもよい。 In the above method, the cell sorting step may be a step of seeding each positive cell in a well of a culture plate, and further including a step of separating and selecting cells in a well that has become confluent by culture. Good.
 上記目的を達成するため、増殖の早いヒト間葉系幹細胞の細胞集団の特徴は、ヒト間葉系幹細胞が含まれる細胞集団から、増殖の早いヒト間葉系幹細胞を分離、選別及び培養し、同分離、選別及び培養した細胞集団においてRor2またはFzd5を発現している細胞(の存在比率)を定量して各細胞集団の合否判定を行い、合格の細胞集団のみを選別することにある。 In order to achieve the above object, the characteristics of a cell population of fast-growing human mesenchymal stem cells are characterized by separating, selecting and culturing fast-growing human mesenchymal stem cells from a cell population containing human mesenchymal stem cells, In the separated, sorted, and cultured cell population, the cells expressing Ror2 or Fzd5 (presence ratio thereof) are quantified, and pass / fail judgment of each cell population is performed, and only the passed cell population is selected.
 同特徴において、ヒト間葉系幹細胞が含まれる細胞集団から、抗LNGFRモノクローナル抗体および抗Thy1モノクローナル抗体で染色した細胞をFCMで解析し、LNGFR Thy1共陽性細胞をセルソーティングし、培養プレートのウェルに同共陽性の各細胞を播種し、培養によりコンフルエントとなったウェルの細胞を分離及び選別した後、前記定量を行ってもよい。 In the same feature, cells stained with an anti-LNGFR monoclonal antibody and an anti-Thy1 monoclonal antibody from a cell population containing human mesenchymal stem cells are analyzed by FCM, and LNGFR Thy1 co-positive cells are cell-sorted into the wells of a culture plate. The quantification may be performed after seeding the cells that are co-positive and separating and sorting the cells in the wells that have become confluent by culture.
 一方、同特徴において、ヒト間葉系幹細胞が含まれる細胞集団から、抗Ror2モノクローナル抗体で染色した細胞をFCMで解析し、Ror2陽性細胞をセルソーティングし、培養プレートのウェルに同陽性の各細胞を播種し、培養によりコンフルエントとなったウェルの細胞を分離及び選別した後、前記定量を行ってもよい。 On the other hand, in the same feature, from a cell population containing human mesenchymal stem cells, cells stained with anti-Ror2 monoclonal antibody are analyzed by FCM, Ror2-positive cells are cell-sorted, and each positive cell is cultured in a well of a culture plate. The above-mentioned quantification may be performed after seeding and separating and sorting cells in a well that has become confluent by culture.
 上記本発明に係る目的を達成するため、本発明に係る新規なモノクローナル抗体は、抗Ror2モノクローナル抗体であり、クローン名が7C9である。また、本発明に係る他の新規なモノクローナル抗体は、抗Fzd5モノクローナル抗体であり、クローン名が6F5である。 In order to achieve the object according to the present invention, the novel monoclonal antibody according to the present invention is an anti-Ror2 monoclonal antibody, and the clone name is 7C9. Another novel monoclonal antibody according to the present invention is an anti-Fzd5 monoclonal antibody, and the clone name is 6F5.
 特定した2つの遺伝子(Fzd5, Ror2:詳細は後述)がコードする蛋白の発現はRECに特異的であり、品質が劣化した細胞集団では発現が認められない。またRECの未分化状態維持に必須の遺伝子であり、1)発現阻害により細胞性能の劣化が誘導される、2)遺伝子の強制発現により未分化状態が延長する、などの結果から単なるバイオマーカーではなく、細胞機能に密接に関与し、細胞性能を担保する有効な指標である。 The expression of the protein encoded by the two specified genes (Fzd5, Ror2: details will be described later) is specific to REC and is not observed in the cell population with degraded quality. It is an essential gene for maintaining the undifferentiated state of REC. 1) Cell performance degradation is induced by inhibition of expression. 2) Undifferentiated state is prolonged by forced expression of gene. Rather, it is closely related to cell function and is an effective index that guarantees cell performance.
 このように、本発明によれば、ヒト間葉系幹細胞の品質評価方法、ヒト間葉系幹細胞の分離、選別及び培養方法、増殖の早いヒト間葉系幹細胞の細胞集団、並びに、増殖の早いヒト間葉系幹細胞を特異的に認識するモノクローナル抗体を提供することが可能となった。 Thus, according to the present invention, human mesenchymal stem cell quality evaluation method, human mesenchymal stem cell isolation, selection and culture method, rapidly proliferating human mesenchymal stem cell population, and rapid proliferation It has become possible to provide monoclonal antibodies that specifically recognize human mesenchymal stem cells.
 本発明の他の目的、構成及び効果については、以下の発明の実施の形態の項から明らかになるであろう。 Other objects, configurations, and effects of the present invention will be apparent from the following embodiments of the present invention.
高品質間葉系幹細胞(REC)の選別、分離、培養及び品質評価工程を示す図である。It is a figure which shows the selection, isolation | separation, culture | cultivation, and quality evaluation process of a high quality mesenchymal stem cell (REC). REC, MEC, SECを様々なパラメータで比較した結果である。It is the result of comparing REC, MEC, and SEC with various parameters. Ror2, Fzd5がREC特異的に発現することを示す図とグラフである。It is a figure and a graph which show that Ror2, and Fzd5 express REC specifically. Fzd5の阻害がRECの細胞性質の劣化を誘導することを示す図とグラフである。FIG. 2 is a graph and graph showing that inhibition of Fzd5 induces degradation of the cellular properties of REC. Fzd5の強制発現がRECの未分化性を誘導することを示す写真とグラフである。It is the photograph and graph which show that forced expression of Fzd5 induces the undifferentiation of REC. 新規に作製した抗Fzd5モノクローナル抗体の染色性を示す写真とグラフである。It is the photograph and graph which show the dyeing | staining property of the newly produced anti- Fzd5 monoclonal antibody. 新規に作製した抗Ror2モノクローナル抗体の染色性を示す写真と図である。It is the photograph and figure which show the dyeing | staining property of the newly produced anti-Ror2 monoclonal antibody. REC特異的抗体を用いた、培養MSCの品質評価を行う模式図である。It is a schematic diagram which performs quality evaluation of culture | cultivation MSC using a REC specific antibody.
 以下、図面を参照しながら、RECの選択、分離及び培養の工程の概要について説明し、続いて、各工程の趣旨と詳細について説明する。本発明では、工程1でRECの選択、分離及び培養を行い、工程2で培養されたRECの評価を行う。工程の組み合わせを表1に例示するが、「プロセス評価欄」で「不可」とされているもの以外は実施しうる。以下、プロセス番号P1,P2のものをまず例示する。 Hereinafter, the outline of the REC selection, separation, and culture process will be described with reference to the drawings, and then the purpose and details of each process will be described. In the present invention, REC is selected, separated and cultured in Step 1, and the REC cultured in Step 2 is evaluated. The combinations of steps are illustrated in Table 1, but can be carried out except for those that are set to “impossible” in the “process evaluation column”. Hereinafter, process numbers P1 and P2 will be exemplified first.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
 [工程1] 図1に単クローン培養法によるREC分離工程を例示する。
1)ヒト骨髄(または脂肪・胎盤絨毛膜)より単核細胞を調製し、骨髄単核細胞を抗LNGFR、抗Thy1で染色する(LNGFR Thy1共陽性細胞をLT細胞とする。)。
2)フローサイトメトリー(FCM、セルソータ)を用い、LNGFR陽性・Thy1陽性細胞を96穴培養プレートにクローンソート(1ウェルに細胞1個ずつ播種すること、表1で「単一」と表記)を行う。
 なお、抗CD106モノクローナル抗体を加え、LNGFR陽性・Thy1陽性かつCD106強陽性の細胞をクローンソートしてもよい。
3)単一細胞培養2週間後に培養プレートを顕微鏡下で撮影し、コンフレントになったウェルを選別し、各ウェルに含まれる細胞をREC(Rapidly Expanding Cells)とする。遅れて増殖しているウェルMEC/SEC(Moderately/Slowly Expanding Cells)は破棄する。
4)RECとして選別した各ウェルから細胞をウェル毎に別々に回収する。1ウェルから回収したRECを1ロットとする。
[Step 1] FIG. 1 illustrates a REC separation step by a monoclonal culture method.
1) Mononuclear cells are prepared from human bone marrow (or fat / placental chorion), and bone marrow mononuclear cells are stained with anti-LNGFR and anti-Thy1 (LNGFR Thy1 co-positive cells are referred to as LT cells).
2) Using flow cytometry (FCM, cell sorter), clone the LNGFR-positive / Thy1-positive cells into a 96-well culture plate (seeding one cell per well, labeled “single” in Table 1) Do.
In addition, an anti-CD106 monoclonal antibody may be added, and LNGFR positive / Thy1 positive and CD106 strong positive cells may be clonal-sorted.
3) After 2 weeks of single cell culture, the culture plate is photographed under a microscope, and confluent wells are selected, and the cells contained in each well are designated as REC (Rapidly Expanding Cells). The well-growing well MEC / SEC (Moderately / Slowly Expanding Cells) is discarded.
4) Collect cells separately from each well selected as REC. One lot of REC collected from one well.
 [工程2] 図1に、さらにRECマーカー(抗Ror2・抗Fzd5)による培養細胞の評価を示す。
1)96穴プレートから回収したRECを各ウェル毎に培養皿または培養フラスコに移入し、コンフレントになるまで培養する(拡大培養)。
2)拡大培養後、全ロットから付着増殖した細胞を回収し、各ロットの一部 (1-3×103個程度)の細胞をとりわけて、RECマーカー(抗Ror2・抗Fzd5)に対するモノクローナル抗体で単一染色を行う。
3)RECマーカー陽性細胞をフローサイトメトリーで解析し、回収細胞におけるRECマーカー陽性細胞の比率を求める(定量的PCRを用いてRor2のmRNA発現を定量してもよいし、顕微鏡により同比率をマニュアルにより求めてもよい)。
4)上記陽性比率が一定値(例えば65%)以上のロット(細胞集団)を合格とする。
5)合格ロットの細胞を凍結バイアルに封入し液体窒素中で保存する。
6)凍結細胞したものを高品質ヒト間葉系幹細胞(製品)とする。
7)ユーザーはバイアルに入った細胞を融解後、培養皿またはフラスコ上で拡大培養を行うことで、最終的に1×1010以上の高純度間葉系幹細胞を安定して使用することが可能である。
[Step 2] FIG. 1 further shows evaluation of cultured cells using a REC marker (anti-Ror2 / anti-Fzd5).
1) REC recovered from the 96-well plate is transferred to each culture dish or culture flask for each well and cultured until confluent (expansion culture).
2) After expansion culture, cells that adhere and proliferate from all lots are collected, and a part of each lot (about 1 to 3 × 10 3 cells), especially monoclonal antibodies against REC markers (anti-Ror2 and anti-Fzd5) Perform single staining with.
3) Analyzing REC marker positive cells by flow cytometry, and determining the ratio of REC marker positive cells in the recovered cells (Ror2 mRNA expression may be quantified using quantitative PCR, or the ratio may be manually determined using a microscope. May be determined by:
4) A lot (cell population) in which the positive ratio is a certain value (for example, 65%) or more is accepted.
5) Enclose the acceptable lot of cells in a frozen vial and store in liquid nitrogen.
6) The frozen cells are used as high-quality human mesenchymal stem cells (product).
7) After thawing the cells in the vial, the user can cultivate the cells on a culture dish or flask and finally use a high-purity mesenchymal stem cell of 1 × 10 10 or more stably. It is.
 上記において、LT細胞をクローンソーティングする代わりに、Ror2陽性細胞をクローンソーティングしてもよい(表1、P6,7)。また、LT細胞、Ror2陽性細胞を選定し、96穴培養プレートの各ウェルに複数個播種してもよい(表1で「複数」と表記、P5,9,14)が、この場合は、クローンソートよりも純度が低下する。なお、コンフレントとは培養容器表面の90%以上を培養細胞が覆っている状態である。また、セミコンフレントとは培養容器表面の70-80%を培養細胞が覆っている状態である。使用する培養器具のサイズおよび種類は細胞の増殖速度に応じ適宜変更可能である。 In the above, Ror2-positive cells may be clone-sorted instead of clone-sorting LT cells (Table 1, P6, 7). Alternatively, LT cells and Ror2-positive cells may be selected and seeded in each well of a 96-well culture plate (indicated as “plurality” in Table 1, P5, 9, 14). Purity is lower than sorting. In addition, the confluent is a state in which the cultured cells cover 90% or more of the surface of the culture container. Semi-confinent is a state in which cultured cells cover 70-80% of the surface of the culture vessel. The size and type of the culture instrument to be used can be appropriately changed according to the cell growth rate.
 上記実施形態では、骨髄を始めとする各組織由来細胞から直接前記細胞集団を調製したが、Ror2陽性細胞でソーティングする場合は、骨髄をはじめとする組織由来細胞を付着培養することにより、前記細胞集団を調製してもよい(表1、P10-15、「付着培養細胞」と表記)。この場合、骨髄単核細胞を10-20%血清+bFGF添加培地(37℃、1-5% CO2)上に播種して約2週間培養し、培養後に出現する繊維芽細胞様の付着細胞(CFU-F)を回収する。細胞集団を調製する工程は、骨髄をコラゲナーゼで処理する工程を包含してもよい。また、同工程は、G-CSF投与後の末梢血から細胞集団を調製するようにしてもよい。 In the above embodiment, the cell population is prepared directly from cells derived from tissues such as bone marrow. However, when sorting with Ror2-positive cells, the cells are obtained by attaching and culturing cells derived from tissues such as bone marrow. Populations may be prepared (Table 1, P10-15, labeled “adherent cultured cells”). In this case, bone marrow mononuclear cells are seeded on a medium containing 10-20% serum + bFGF (37 ° C., 1-5% CO 2 ) and cultured for about 2 weeks, and fibroblast-like adherent cells appearing after the culture. Collect (CFU-F). The step of preparing the cell population may include a step of treating the bone marrow with collagenase. In the step, a cell population may be prepared from peripheral blood after administration of G-CSF.
 なお、出荷前の評価(工程2の2),3))は必ずしも必要ではなく、表1プロセスP15の如く、付着培養細胞を利用し、Ror2陽性のものをFMCでソーティングにより分離し、必要に応じて拡大培養し、これを[工程2]の5)以降の処理により出荷してもよい。 In addition, evaluation before shipment (2) and 3)) before shipment is not always necessary. As shown in Table 1 Process P15, adherent cultured cells are used, and Ror2-positive cells are separated by FMC and sorted. Accordingly, the cells may be expanded and then shipped by the process after 5) of [Step 2].
 ここで、図2を参照しながら、RECとMEC/SECの細胞性能を比較する。REC, MEC, SECの細胞性能を様々なパラメータで比較した結果を示す。
同図Aは、ヒト骨髄単核細胞をLNGFRおよびThy1に対する抗体で染色後、FCM解析を行った結果である。楕円で囲まれた部分がLT細胞である。
同図Bは、LT細胞を96穴プレートに単一細胞分離(クローンソート)することの模式図である。
同図Cは、単一細胞培養後の細胞数を定期的に計測した結果を示すグラフである。RECはMEC/SECと比較し増殖速度が速く、約2週間で0.5-1×104個まで増殖する。0.5-1×104個は96穴プレートのウェルがコンフレントになる数である。
同図Dは、REC・MEC・SECを骨・脂肪へ分化誘導した後、骨・脂肪細胞に特異的な遺伝子発現を定量PCRにて計測した結果を示す。RECはMEC, SECと比較し、特に脂肪分化能が高いことが示された。
同図Eは、REC・MEC・SECを再度96穴プレートにクローンソート後、2次コロニーを形成したウェルの数を比較した結果のグラフである。2次コロニーの形成は未分化状態の目安とされる自己複製能の指標である。RECの約33%が2次コロニーを形成するのに対し、MEC/SECはごく僅かなコロニーしか形成されない。
同図Fは、Luc(ルシフェラーゼ)遺伝子発現ベクターを導入した以下の細胞集団、WBM(通常法で得たMSC、WBMはWhole Bone Marrowの略称である)、REC、MEC/SEC、および陰性対照群としてルシフェラーゼで標識していないWBM(Luc(-) Cultured MSC)をそれぞれ免疫不全マウスに対し経静脈的に投与後に、Lucの基質であるルシフェリンを腹腔内投与し、ルシフェラーゼの発光を体外から検出できる装置(IVIS)を用いて、移植24時間後に観察した結果を示す。上段)グラフは各マウスにおけるLuc発光量を数値化し、WBM ・MSC移植した群を100%とした時の他の細胞を移植したマウスの発光比率(%)をプロットしたものである。下段)画像は各群のレシピエントマウスにおけるLucの発光をイメージ化したものである。いずれの結果からも、RECを移植したマウスは肺での発光量が極端に低いことから、RECは肺毛細血管にほとんどトラップされないのに対し、MEC/SECはWBM(通常上で得た培養MSC)とほぼ同等に補足され、肺中にとどまっていることがわかる。
 以上の結果を総合すると、RECは増殖能・分化能・遊走能ともに優れた細胞集団であり、特に後述する難治疾患への全身投与が可能という点で、新鮮骨髄中のMSCに匹敵する遊走性を維持していることがポイントとなる。
Here, the cell performance of REC and MEC / SEC will be compared with reference to FIG. The result of having compared the cell performance of REC, MEC, and SEC with various parameters is shown.
FIG. A shows the results of FCM analysis after staining human bone marrow mononuclear cells with antibodies against LNGFR and Thy1. The portion surrounded by the ellipse is the LT cell.
FIG. B is a schematic diagram of single cell separation (clone sorting) of LT cells in a 96-well plate.
FIG. C is a graph showing the results of periodically measuring the number of cells after single cell culture. REC has a faster growth rate than MEC / SEC and grows to 0.5-1 × 10 4 cells in about 2 weeks. 0.5-1 × 10 4 is the number at which the wells of the 96-well plate become confluent.
FIG. D shows the results of quantitative expression PCR measuring gene expression specific to bone and fat cells after inducing differentiation of REC, MEC and SEC into bone and fat. REC was shown to have particularly high fat differentiation ability compared to MEC and SEC.
FIG. E is a graph showing the results of comparing the number of wells in which secondary colonies were formed after REC / MEC / SEC were again cloned into a 96-well plate. The formation of secondary colonies is an index of self-replication ability, which is a measure of the undifferentiated state. About 33% of RECs form secondary colonies, whereas MEC / SEC forms very few colonies.
FIG. F shows the following cell population introduced with a Luc (luciferase) gene expression vector, WBM (MSC obtained by a conventional method, WBM is an abbreviation for Whole Bone Marrow), REC, MEC / SEC, and negative control group As the luciferase luminescence, luciferase can be detected from outside the body after intravenous administration of WBM (Luc (-) Cultured MSC) unlabeled with luciferase to each immunodeficient mouse. The result observed 24 hours after transplantation using the device (IVIS) is shown. The upper graph is a graph in which the amount of Luc luminescence in each mouse is digitized, and the luminescence ratio (%) of mice transplanted with other cells when the WBM / MSC transplanted group is taken as 100% is plotted. (Lower) The image is an image of Luc luminescence in each group of recipient mice. From both results, REC-transplanted mice have extremely low light emission in the lung, so REC is hardly trapped in lung capillaries, whereas MEC / SEC is WBM (cultured MSC obtained above). ) Is supplemented almost equivalently, and remains in the lungs.
To sum up the above results, REC is a cell population with excellent proliferation ability, differentiation ability, and migration ability, and especially migratory ability comparable to MSC in fresh bone marrow in that it can be systemically administered to intractable diseases described later. The point is to maintain.
 以上を含め、発明者らの実験によれば、RECは通常MSCと比較し以下の特徴を持つ。
1.形態的に極めて均一な細胞集団である
2.細胞老化が見られない
3.分裂速度が早く、未分化性を維持したまま培養増幅が可能
4.分化能が高く骨・脂肪へ分化させやすい細胞集団
5.遊走能を維持している
 RECはヒトMSCのうち最も未分化な細胞集団であり、骨髄中のMSCに最も近い性質を持つ。またMEC/SECあるいは通常法で得たMSCと比較し、高い分化・増殖・遊走能にすぐれた、新鮮かつ変異の少ない、細胞性能が保証された細胞集団である。
Including the above, according to experiments by the inventors, REC has the following characteristics compared to normal MSC.
1. 1. Morphologically extremely uniform cell population 2. Cell senescence is not observed. 3. Amplification of culture is possible with high division rate and maintaining undifferentiation. 4. Cell population with high differentiation potential and easy to differentiate into bone and fat REC, which maintains its migration ability, is the most undifferentiated cell population among human MSCs, and has properties closest to those in bone marrow. Compared to MSC obtained by MEC / SEC or conventional methods, it is a cell population with excellent cell differentiation, proliferation, and migration ability, freshness, low mutation, and guaranteed cell performance.
 続いて、図3を参照しながら、未分化MSC(REC)特異的遺伝子Ror2,Fzd5の同定について説明する。
 REC,MEC,SECそれぞれで発現する遺伝子の発現レベルをDNAアレイ法により比較し、Wnt(ウイント)受容体の一つであるFzd5およびその共受容体であるRor2がREC特異的であることを確認した。
 同図Aは、REC,MEC,SECそれぞれにおける定量的PCRによるRor2 mRNAの発現比較である。
 同図BはREC,MEC,SECそれぞれにおける定量的PCRによるFzd5 mRNAの発現比較である。
 同図Cはウエスタンブロッティング法によるFzd5蛋白発現の比較である。
 同図Dは、蛍光免疫染色法によるFzd5蛋白の細胞内局在比較写真である。
 以上、複数の解析方法で評価した結果、Fzd5とRor2の発現はREC特異的であることが確認できた。従ってFzd5およびRor2それぞれの発現を検出し定量化すれば、RECの細胞品質評価の指標として有効と考えられる。また、新規に作製した抗Fzd5モノクローナル抗体と抗Ror2モノクローナル抗体は、フローサイトメトリー、ウエスタンブロッティング、蛍光免疫染色いずれの手法においても対象とするタンパク抗原の検出と定量化が可能である。
Next, identification of undifferentiated MSC (REC) specific genes Ror2 and Fzd5 will be described with reference to FIG.
The expression levels of genes expressed in REC, MEC and SEC are compared by DNA array method, and it is confirmed that Fzd5, one of Wnt receptors, and Ror2, its co-receptor, are REC-specific. did.
Fig. A is a comparison of Ror2 mRNA expression by quantitative PCR in each of REC, MEC, and SEC.
FIG. B is a comparison of Fzd5 mRNA expression by quantitative PCR in each of REC, MEC, and SEC.
FIG. C is a comparison of Fzd5 protein expression by Western blotting.
FIG. D is a photograph comparing the intracellular localization of Fzd5 protein by fluorescent immunostaining.
As described above, as a result of evaluation by a plurality of analysis methods, it was confirmed that the expression of Fzd5 and Ror2 is REC-specific. Therefore, if the expression of each of Fzd5 and Ror2 is detected and quantified, it is considered effective as an index for evaluating the cell quality of REC. In addition, the newly produced anti-Fzd5 monoclonal antibody and anti-Ror2 monoclonal antibody can detect and quantify the target protein antigen in any of flow cytometry, western blotting, and fluorescent immunostaining.
 次に、図4を参照しながら、Fzd5のloss of functionによる細胞老化誘導について検討する。
 RNAインタフェレース法は対象とするmRNAに対し相補的な配列を持つ短いRNA(shRNA)を細胞内に導入し、対象とするmRNAを破壊することで目的遺伝子の機能を調べる手法である。Fzd5に対し相補的な配列を持つshRNA(shFZD5)によりFzd5 mRNAを破壊した場合のRECの細胞性質を対照群(shCTRL:Fzd5に対し相補的ではないランダム配列のshRNA)と比較した一連の実験結果を示す。
 同図Aは、shFZD5またはshCTRLをそれぞれRECに導入した後、Fzd5 のmRNA量を定量的PCR法にて定量化したグラフである。対照群(shCTRL、ショートヘアピンコントロール)におけるFzd5 mRNA量を100とした場合、shFZD5を強制発現したRECではFzd5のmRNA量が約40%に低下していた。
 同図Bは、shFZD5またはshCTRLをそれぞれRECに導入した後、対照群の細胞数を1とした時のshFZD5強制発現群の細胞数を縦軸、shRNA導入後の日数を横軸にプロットしたグラフである。shFZD5を発現したRECは対照群と比較し、細胞数の急激な減少が認められ、Rzd5の阻害により増殖能の低下が誘導されることが示唆された。
 同図Cは、shFZD5またはshCTRLをそれぞれRECに導入後、脂肪細胞へ分化誘導し、培養14日目にOil-Red-Oで脂肪滴を染色した画像である。対照群と比較しFzd5を阻害すると脂肪分化能の低下が認められた。
 同図Dは、細胞老化の指標であるSA-β-gal活性は基質であるX-galを加えると青色に染色され検出できる。shFZD5またはshCTRLをそれぞれRECに導入後、x-gal染色を行った画像および各細胞集団におけるSA-β-gal活性を持つ細胞頻度をプロットしたグラフを示す。
 同図Eは、細胞老化の指標であるp16(INK4a)のmRNA量を定量的PCR法により定量化したグラフである。対照群を100とした場合、shFZD5導入RECにおけるp16 mRNA量は約300であり、Fzd5の発現阻害により細胞老化が誘導されることが示された。
 同図Fは、shFZD5またはshCTRLをそれぞれRECに導入後、各細胞集団に対し抗F-actin抗体にて細胞内染色を行いStress Fiberの形成を観察した画像、および同細胞集団それぞれに含まれる各細胞の面積(細胞サイズ)の平均値をプロットしたグラフを示す。
 以上の結果から、RECにおけるFzd5の機能を阻害することにより、増殖能の低下、分化能の低下、細胞老化の誘導、Stress fiber形成による遊走性の低下と細胞サイズの増大が誘導され、MEC/SECと同じ性状に変化することから、Fzd5は単なるバイオマーカーではなく、RECの細胞性能の維持を担保する機能分子と考えられる。
Next, cell aging induction by Fzd5 loss of function will be examined with reference to FIG.
The RNA interlacing method is a method for examining the function of a target gene by introducing a short RNA (shRNA) having a sequence complementary to the target mRNA into the cell and destroying the target mRNA. Results of a series of experiments comparing the cellular properties of REC when Fzd5 mRNA was disrupted by shRNA having a sequence complementary to Fzd5 (shFZD5) with the control group (shCTRL: shRNA having a random sequence not complementary to Fzd5) Indicates.
Fig. A is a graph in which the amount of Fzd5 mRNA was quantified by quantitative PCR after introducing shFZD5 or shCTRL into REC. When the amount of Fzd5 mRNA in the control group (shCTRL, short hairpin control) was 100, the amount of Fzd5 mRNA was reduced to about 40% in the REC in which shFZD5 was forcibly expressed.
Fig. B is a graph in which, after introducing shFZD5 or shCTRL into REC, the number of cells in the shFZD5 forced expression group when the number of cells in the control group is 1 is plotted on the vertical axis, and the number of days after shRNA introduction is plotted on the horizontal axis. It is. Compared with the control group, REC expressing shFZD5 showed a rapid decrease in the number of cells, suggesting that the inhibition of Rzd5 induces a decrease in proliferation ability.
Fig. C is an image of shFZD5 or shCTRL introduced into REC, induced to differentiate into adipocytes, and stained with Oil-Red-O on the 14th day of culture. When Fzd5 was inhibited as compared with the control group, a decrease in fat differentiation ability was observed.
In FIG. D, SA-β-gal activity, which is an indicator of cell senescence, can be detected by staining with blue when the substrate X-gal is added. An image in which shFZD5 or shCTRL is introduced into REC and then stained with x-gal and a graph plotting the frequency of cells having SA-β-gal activity in each cell population are shown.
FIG. E is a graph in which the amount of mRNA of p16 (INK4a), which is an indicator of cell senescence, is quantified by a quantitative PCR method. When the control group was 100, the amount of p16 mRNA in shFZD5-introduced REC was about 300, indicating that cell senescence was induced by inhibition of Fzd5 expression.
Fig. F shows images obtained by introducing shFZD5 or shCTRL into each REC and then staining each cell population with intracellular anti-F-actin antibody to observe the formation of Stress Fiber, and each of the cell populations. The graph which plotted the average value of the area (cell size) of a cell is shown.
From the above results, inhibition of the function of Fzd5 in REC leads to a decrease in proliferation ability, a decrease in differentiation ability, induction of cell senescence, a decrease in migration due to stress fiber formation and an increase in cell size, and MEC / Since it changes to the same property as SEC, Fzd5 is not a mere biomarker, but is considered to be a functional molecule that ensures the maintenance of the cell performance of REC.
 次に、図5を参照しながら、Fzd5の gain of functionによる長期増殖能維持について考察する。
 Fzd5の全長cDNAをRECに強制発現することでFzd5 mRNAを恒常的に発現させたことによる細胞機能への影響を確認した。発現ベクターはFzd5 cDNAと蛍光蛋白GFP(Green Fluorescent Protein、緑色蛍光タンパク質)がタンデムに連なっており、Fzd5遺伝子導入細胞はGFPを同時に発現しているため蛍光顕微鏡を用いて導入した遺伝子の発現が確認できる。
 同図Aは、蛍光顕微鏡によるGFP発現細胞の形態観察写真である。Fzd5 cDNAとGFPを導入した細胞集団(Fzd5)とGFP遺伝子のみを導入した対照群(CTRL)の遺伝子導入後28日目の細胞形態を撮影した画像である。
 対照群では図中矢印で示した細胞老化の特徴であるサイズの大きい多極性細胞が多数出現しているのに対し、Fzd5発現RECはほぼ全てが細胞質の小さい双極性の形態を維持していた。
 同図Bは、対照群の細胞数を1とした時のFzd5発現RECの細胞数を縦軸、遺伝子導入後の日数を横軸にプロットしたグラフである。対照群と比較し、Fzd5を強制的に発現させたRECでは増殖能が長期的に維持されていた。
 以上の結果から、Fzd5を介したWntシグナル刺激により、未分化性を維持した状態で長期的な培養増幅が可能になることが見込まれる。
Next, the long-term proliferative ability maintenance by the gain of function of Fzd5 is considered, referring to FIG.
The influence on the cell function due to the constant expression of Fzd5 mRNA was confirmed by forcibly expressing the full length cDNA of Fzd5 in REC. The expression vector consists of Fzd5 cDNA and fluorescent protein GFP (Green Fluorescent Protein) in tandem, and Fzd5 gene-introduced cells simultaneously express GFP, confirming the expression of the introduced gene using a fluorescence microscope. it can.
FIG. 5A is a morphological observation photograph of GFP-expressing cells using a fluorescence microscope. It is the image which image | photographed the cell form of the 28th day after gene introduction of the cell group (Fzd5) which introduce | transduced Fzd5 cDNA and GFP, and the control group (CTRL) which introduce | transduced only the GFP gene.
In the control group, many large-polarized multipolar cells, which are characteristic of cell senescence indicated by arrows in the figure, appeared, whereas almost all Fzd5-expressing RECs maintained a cytoplasmic bipolar bipolar morphology. .
FIG. B is a graph in which the number of Fzd5-expressing REC cells is plotted on the vertical axis and the number of days after gene introduction is plotted on the horizontal axis when the number of cells in the control group is 1. Compared with the control group, the growth ability of REC in which Fzd5 was forcibly expressed was maintained for a long time.
From the above results, it is expected that long-term culture amplification will be possible with Wnt signal stimulation via Fzd5 while maintaining undifferentiation.
 続いて、図6を参照しながら、ヒトFzd5に対する新規モノクローナル抗体の作製について説明する。
 ヒトFzd5抗原の細胞外領域を免疫原とし、ホストマウスを免疫後、常法に従いハイブリドーマを作製し、Fzd5遺伝子を発現させたBa/F3細胞でスクリーニングを行うことで、新規の抗Fzd5モノクローナル抗体(クローン名:6F5)を得た。
 本抗体を用い様々な手法でFzd5蛋白が検出できるか否かの確認を行った。
 同図Aは、Fzd5の細胞外領域を強制発現させたBa/F3細胞に対し、Biotin標識した6F5抗体で染色後、ストレプトアビジン(SAV)-PEで蛍光標識しフローサイトメトリーで解析を行い、PEの蛍光強度を横軸にプロットしたヒストグラムを示す。図中のグレーで示したヒストグラムは一次抗体としてアイソタイプコントロールを加えた陰性コントロール、白抜きのヒストグラムは6F5で染色したサンプルのPE蛍光強度である。図中の横バーで示した領域がFzd5陽性細胞領域であり、数値は陽性率(%)を表す。
 同図Bは、RECの異なる3クローンより細胞内蛋白を調製し、6F5を一次抗体としてFzd5蛋白を検出したウエスタンブロッティングの結果を示す。陰性コントロールとして、サル腎臓由来の細胞株COS7より調製した細胞内蛋白を用いた。
 同図Cは、REC細胞に対し6F5-Biotinを一次抗体として染色後、ストレプトアビジン(SAV)-Alexa555にて蛍光ラベル後に蛍光顕微鏡で観察、撮影した画像である。抗Fzd5抗体(6F5)はフローサイトメトリー、ウエスタンブロッティング、蛍光免疫染色の全てに利用可能であった。
Subsequently, the production of a novel monoclonal antibody against human Fzd5 will be described with reference to FIG.
By using the extracellular region of human Fzd5 antigen as an immunogen and immunizing a host mouse, a hybridoma is prepared according to a conventional method, and screening is performed on Ba / F3 cells expressing the Fzd5 gene, whereby a novel anti-Fzd5 monoclonal antibody ( Clone name: 6F5) was obtained.
It was confirmed whether Fzd5 protein could be detected by various techniques using this antibody.
In Fig. A, Ba / F3 cells forcibly expressing the extracellular region of Fzd5 were stained with 6F5 antibody labeled with Biotin, then fluorescently labeled with streptavidin (SAV) -PE, and analyzed by flow cytometry. The histogram which plotted the fluorescence intensity of PE on the horizontal axis is shown. The histogram shown in gray in the figure is the negative control with the isotype control added as the primary antibody, and the white histogram is the PE fluorescence intensity of the sample stained with 6F5. The region indicated by the horizontal bar in the figure is the Fzd5-positive cell region, and the numerical value represents the positive rate (%).
FIG. B shows the results of Western blotting in which intracellular proteins were prepared from 3 clones with different RECs, and Fzd5 protein was detected using 6F5 as a primary antibody. As a negative control, an intracellular protein prepared from monkey kidney-derived cell line COS7 was used.
FIG. C is an image of REC cells stained with 6F5-Biotin as a primary antibody, observed with a fluorescent microscope after streptavidin (SAV) -Alexa555 and labeled with a fluorescent microscope. Anti-Fzd5 antibody (6F5) was available for all of flow cytometry, Western blotting, and fluorescent immunostaining.
 次に、図7を参照しながら、ヒトRor2抗原に対する新規モノクローナル抗体の作製について説明する。
 ヒトRor2抗原を免疫原とし、新規に抗Ror2抗体(クローン名:7C9)を作製した。同クローンを用い、
 同図AはRECに対し、1次抗体として7C9-Biotinで染色後、SAV-PEで蛍光ラベルし、フローサイトメトリーを用いてPE蛍光の検出を行った結果を示す。1次抗体としてアイソタイプコントロール抗体を加えたサンプルを陰性コントロールとする。縦軸にFITC蛍光(染色していないため全て陰性)、横軸にPE蛍光をプロットした2次元ドットプロットである。陰性コントロール群ではPE蛍光を発する細胞がほぼ含まれない領域(図中の台形で囲まれた部分:0.011%)がClone7C9で染色したサンプルにおいてPE蛍光を発現している細胞集団(69.3%)であった。
 同図Bは、RECに対し7C9-Biotinを1次抗体として免疫染色を行い、Streptavidin-Alexa488で蛍光標識後にRor2蛋白の発現を蛍光顕微鏡にて観察・撮影した画像である。RECの大部分がRor2蛋白を発現していることが確認された。
 同図Cは、新鮮骨髄細胞に対しLNGFR-APC, Thy1-FITC, Ror2-PE(それぞれの抗原に対するモノクローナル抗体)による3重染色を行い、フローサイトメトリー解析を行った結果を示す。左はLNGFRの発現を縦軸に、Thy1の発現を横軸にプロットした図であり、四角枠で囲まれた部分はヒトMSCが高頻度に含まれるLNGFR Thy1共陽性細胞集団である。右2つの図はLNGFR Thy1共陽性細胞集団のみを抽出後、横軸を細胞の大きさの指標であるFSC、縦軸にSAV-PEで標識した抗Ror2-Biotin抗体(7C9)のPE蛍光をプロットした図である。四角枠で囲まれた部分は陰性コントロールを元に設定したRor2陽性領域であり、数値は陽性率(%)を表す。Clone7C9を用いた場合、LNGFR Thy1共陽性細胞の92.3%がRor2陽性であり、LNGFR Thy1に代わるMCSの選別マーカーとして用いることが可能である。
 新規に作製した抗Ror2モノクローナル抗体を用いることで、フローサイトメトリー及び蛍光免疫染色によりRor2蛋白の検出と定量化が可能である(図7A,B)。
 さらに新規に作製した抗Ror2は骨髄中に含まれるMSCのマーカーとしても利用可能である(図7C)。
Next, the production of a novel monoclonal antibody against human Ror2 antigen will be described with reference to FIG.
A novel anti-Ror2 antibody (clone name: 7C9) was prepared using human Ror2 antigen as an immunogen. Using the same clone,
Fig. A shows the results of staining REC with 7C9-Biotin as a primary antibody, fluorescently labeling with SAV-PE, and detecting PE fluorescence using flow cytometry. A sample to which an isotype control antibody is added as a primary antibody is used as a negative control. It is a two-dimensional dot plot in which the vertical axis is FITC fluorescence (all negative because it is not stained), and the horizontal axis is PE fluorescence. In the negative control group, a cell population (69.) expressing PE fluorescence in a sample stained with Clone7C9 in a region almost free of cells that emit PE fluorescence (portion surrounded by a trapezoid in the figure: 0.011%). 3%).
FIG. B is an image obtained by performing immunostaining on REC using 7C9-Biotin as a primary antibody, followed by fluorescence labeling with Streptavidin-Alexa488, and observing and photographing the expression of Ror2 protein with a fluorescence microscope. It was confirmed that most of REC expressed Ror2 protein.
FIG. 3C shows the results of flow cytometry analysis of fresh bone marrow cells by triple staining with LNGFR-APC, Thy1-FITC, Ror2-PE (monoclonal antibodies against each antigen). On the left is a diagram in which LNGFR expression is plotted on the vertical axis and Thy1 expression is plotted on the horizontal axis, and the portion surrounded by a square frame is a LNGFR Thy1 co-positive cell population containing human MSCs at a high frequency. The two figures on the right show only the LNGFR Thy1 co-positive cell population, and the horizontal axis shows the FSC, which is an indicator of cell size, and the vertical axis shows the PE fluorescence of the anti-Ror2-Biotin antibody (7C9) labeled with SAV-PE. FIG. The portion surrounded by a square frame is a Ror2 positive region set based on the negative control, and the numerical value represents the positive rate (%). When Clone7C9 is used, 92.3% of LNGFR Thy1 co-positive cells are Ror2 positive and can be used as a selection marker for MCS in place of LNGFR Thy1.
By using a newly prepared anti-Ror2 monoclonal antibody, Ror2 protein can be detected and quantified by flow cytometry and fluorescent immunostaining (FIGS. 7A and 7B).
Furthermore, the newly produced anti-Ror2 can be used as a marker for MSC contained in the bone marrow (FIG. 7C).
 次に、図8を参照しながら、抗Ror2抗体または抗Fzd5抗体を用いた、細胞品質の評価手順について説明する。
 通常法である付着培養を行ったヒトMSC(または継代培養を行ったREC)を回収し、REC特異的なモノクローナル抗体(抗Ror2抗体または抗Fzd5抗体)で染色する。
 フローサイトメトリーにて陽性細胞の頻度(含有%)を計測する。その代わりに、蛍光顕微鏡下で陽性細胞の頻度(含有%)を計測してもよい。これらの計測により、その細胞集団にどれだけのRECが含まれているかを定量化することができるため、対象とするMSCがどの程度の分化・増殖・遊走能を持つか、細胞品質を評価することが可能である。発明者らの実験によれば、RECのRor2陽性率は5ロットで72%±8.9%であった。よって、例えば、最低値である63%以上や、65%以上を合否判定の基準値としてもよい。
Next, a procedure for evaluating cell quality using an anti-Ror2 antibody or an anti-Fzd5 antibody will be described with reference to FIG.
Human MSCs (or subcultured RECs) subjected to adherent culture, which is a conventional method, are collected and stained with a REC-specific monoclonal antibody (anti-Ror2 antibody or anti-Fzd5 antibody).
The frequency (% content) of positive cells is measured by flow cytometry. Instead, the frequency (content%) of positive cells may be measured under a fluorescence microscope. By these measurements, it is possible to quantify how much REC is contained in the cell population, so that the cell quality is evaluated as to how much differentiation / proliferation / migration ability the target MSC has. It is possible. According to the inventors' experiment, the Ror2 positive rate of REC was 72% ± 8.9% in 5 lots. Therefore, for example, the minimum value of 63% or more or 65% or more may be used as the reference value for the pass / fail determination.
 本発明によれば、全身性疾患への治療に利用することができるヒト間葉系幹細胞を効率良く分離培養する技術を提供すること、および得られた細胞集団が移植に適しているか、薬効性を示すかの基準となる品質評価を行うことが可能になった。 
 新規に作製した高純度間葉系幹細胞に特化した染色性を示すモノクローナル抗体のうち、細胞分離に適した候補はナノ磁気微粒子と結合させることによって、間葉系幹細胞分離用試薬として製品化できる。また、分離した間葉系幹細胞の品質を検定するための細胞評価用の試薬として、蛍光物質結合抗体、細胞染色用試薬が実用化できる。
 間葉系幹細胞は、従来行われてきたバイオマテリアルの材料として、あるいはその多分化能を生かし、重症筋無力症、慢性リウマチ症等への投与、さらには脊髄損傷、心・血管、慢性肝不全を始めとする重度の疾患治療に対する細胞治療を行う際に組織の場(ニッシェ)を整える支持細胞として共移植するなど、様々な応用が期待される。特に、遊走性を維持しているRECを用いることにより、これまで治療法が存在しなかった低フォスファターゼ症をはじめとする全身性骨・軟骨疾患等の代謝性疾患、GVHDの治療など経静脈的に投与する必要のある全ての疾患に適用すれば、これまでにない治療効果が見込まれる。
According to the present invention, it is possible to provide a technique for efficiently separating and culturing human mesenchymal stem cells that can be used for the treatment of systemic diseases, and whether the obtained cell population is suitable for transplantation, or a medicinal effect. It is now possible to perform quality evaluations that serve as the basis for indicating
Among newly-prepared monoclonal antibodies that exhibit staining properties specialized for high-purity mesenchymal stem cells, candidates suitable for cell separation can be commercialized as reagents for separating mesenchymal stem cells by binding to nanomagnetic particles. . In addition, fluorescent substance-bound antibodies and cell staining reagents can be put to practical use as cell evaluation reagents for examining the quality of the separated mesenchymal stem cells.
Mesenchymal stem cells are used for biomaterials that have been used in the past, or by taking advantage of their multipotency, administration to myasthenia gravis, chronic rheumatism, etc., spinal cord injury, cardiovascular, chronic liver failure Various applications are expected, such as co-transplantation as supporting cells to prepare the tissue (niche) when performing cell therapy for severe disease treatment such as. In particular, by using REC that maintains migratory properties, metabolic diseases such as systemic bone and cartilage diseases such as hypophosphatasia, for which treatment has not been available, and intravenous treatment such as treatment of GVHD If it is applied to all diseases that need to be administered, the therapeutic effect is unprecedented.

Claims (20)

  1. ヒト間葉系幹細胞の品質評価方法であって、
    ヒト間葉系幹細胞が含まれる細胞集団から、増殖の早いヒト間葉系幹細胞を分離、選別及び培養し、
    同分離、選別及び培養した細胞集団においてRor2またはFzd5を発現している細胞の存在比率を定量して各細胞集団の合否判定を行うヒト間葉系幹細胞の品質評価方法。
    A method for evaluating the quality of human mesenchymal stem cells,
    Separating, selecting and culturing fast-growing human mesenchymal stem cells from a cell population containing human mesenchymal stem cells;
    A method for evaluating the quality of human mesenchymal stem cells, wherein the proportion of cells expressing Ror2 or Fzd5 in the separated, sorted and cultured cell populations is quantified to determine pass / fail of each cell population.
  2. 抗Ror2モノクロナール抗体または抗Fzd5モノクロナール抗体を用いてRor2またはFzd5を発現している細胞を定量する請求項1に記載のヒト間葉系幹細胞の品質評価方法。 The method for evaluating the quality of human mesenchymal stem cells according to claim 1, wherein cells expressing Ror2 or Fzd5 are quantified using an anti-Ror2 monoclonal antibody or an anti-Fzd5 monoclonal antibody.
  3. 定量的PCRを用いてRor2のmRNAを発現している細胞を定量する請求項2に記載のヒト間葉系幹細胞の品質評価方法。 The method for evaluating the quality of human mesenchymal stem cells according to claim 2, wherein the cells expressing Ror2 mRNA are quantified using quantitative PCR.
  4. 免疫染色によりRor2またはFzd5を発現している細胞を定量する請求項2に記載のヒト間葉系幹細胞の品質評価方法。 The method for evaluating the quality of human mesenchymal stem cells according to claim 2, wherein cells expressing Ror2 or Fzd5 are quantified by immunostaining.
  5. ヒト間葉系幹細胞の分離、選別及び培養方法であって、
    ヒト間葉系幹細胞が含まれる細胞集団から、増殖の早いヒト間葉系幹細胞を分離、選別及び培養し、
    同分離、選別及び培養した細胞集団においてRor2またはFzd5を発現している細胞の存在比率を定量して各細胞集団の合否判定を行い、合格の細胞集団のみを選別するヒト間葉系幹細胞の分離、選別及び培養方法。
    A method for isolating, selecting and culturing human mesenchymal stem cells,
    Separating, selecting and culturing fast-growing human mesenchymal stem cells from a cell population containing human mesenchymal stem cells;
    Separation of human mesenchymal stem cells by quantifying the abundance ratio of cells expressing Ror2 or Fzd5 in the separated, sorted and cultured cell population, and determining pass / fail of each cell population, and selecting only successful cell populations Sorting and culturing method.
  6. 抗Ror2モノクロナール抗体または抗Fzd5モノクロナール抗体を用いてRor2またはFzd5を発現している細胞を定量する請求項5に記載のヒト間葉系幹細胞の分離、選別及び培養方法。 6. The method for isolating, selecting and culturing human mesenchymal stem cells according to claim 5, wherein cells expressing Ror2 or Fzd5 are quantified using an anti-Ror2 monoclonal antibody or an anti-Fzd5 monoclonal antibody.
  7. 定量的PCRを用いてRor2のmRNAを発現している細胞を定量する請求項6に記載のヒト間葉系幹細胞の分離、選別及び培養方法。 The method for isolating, selecting and culturing human mesenchymal stem cells according to claim 6, wherein cells expressing Ror2 mRNA are quantified using quantitative PCR.
  8. 免疫染色によりRor2またはFzd5を発現している細胞を定量する請求項6に記載のヒト間葉系幹細胞の分離、選別及び培養方法。 The method for isolating, selecting and culturing human mesenchymal stem cells according to claim 6, wherein cells expressing Ror2 or Fzd5 are quantified by immunostaining.
  9. 前記増殖の早いヒト間葉系幹細胞として分離、選別及び培養する工程が、ヒト間葉系幹細胞が含まれる細胞集団から、抗LNGFRモノクロナール抗体および抗Thy1モノクロナール抗体で染色した細胞をフローサイトメトリー(以下、「FCM」)で解析し、LNGFR Thy1共陽性細胞をセルソーティングする工程を含んでいる請求項5に記載のヒト間葉系幹細胞の分離、選別及び培養方法。 The step of separating, selecting, and culturing as a rapidly proliferating human mesenchymal stem cell comprises flow cytometry of cells stained with an anti-LNGFR monoclonal antibody and an anti-Thy1 monoclonal antibody from a cell population containing human mesenchymal stem cells. The method for separating, selecting and culturing human mesenchymal stem cells according to claim 5, comprising a step of cell sorting using LFCFR Thy1 co-positive cells analyzed by (hereinafter “FCM”).
  10. 前記増殖の早いヒト間葉系幹細胞として分離、選別及び培養する工程が、ヒト間葉系幹細胞が含まれる細胞集団から、抗Ror2モノクロナール抗体で染色した細胞をFCMで解析し、Ror2陽性細胞をセルソーティングする工程を含んでいる請求項5に記載のヒト間葉系幹細胞の分離、選別及び培養方法。 The step of isolating, selecting and culturing as fast-growing human mesenchymal stem cells comprises analyzing cells stained with anti-Ror2 monoclonal antibody from a cell population containing human mesenchymal stem cells by FCM, and analyzing Ror2-positive cells. 6. The method for separating, selecting and culturing human mesenchymal stem cells according to claim 5, comprising a step of cell sorting.
  11. 骨髄、その他の各組織由来細胞から直接前記細胞集団を調製する工程を包含する請求項9または10記載のヒト間葉系幹細胞の分離、選別及び培養方法。 The method for separating, selecting and culturing human mesenchymal stem cells according to claim 9 or 10, comprising a step of directly preparing the cell population from bone marrow and other tissue-derived cells.
  12. 骨髄、その他の各組織由来細胞を付着培養することにより、前記細胞集団を調製する工程を包含する請求項10記載のヒト間葉系幹細胞の分離、選別及び培養方法。 The method for separating, selecting and culturing human mesenchymal stem cells according to claim 10, comprising the step of preparing the cell population by adherent culture of bone marrow and other tissue-derived cells.
  13. 前記セルソーティングする工程が、培養プレートのウェルに陽性の各細胞を播種するものであり、その後さらに、培養によりコンフルエントとなったウェルの細胞を分離及び選別する工程を包含する請求項9または10記載のヒト間葉系幹細胞の分離、選別及び培養方法。 11. The cell sorting step includes seeding each positive cell in a well of a culture plate, and further comprising a step of further separating and selecting cells in a well that has become confluent by culture. Of separating, selecting and culturing human mesenchymal stem cells.
  14. ヒト間葉系幹細胞が含まれる細胞集団から、増殖の早いヒト間葉系幹細胞を分離、選別及び培養し、
    同分離、選別及び培養した細胞集団においてRor2またはFzd5を発現している細胞の存在比率を定量して各細胞集団の合否判定を行い、合格の細胞集団のみを選別した増殖の早いヒト間葉系幹細胞の細胞集団。
    Separating, selecting and culturing fast-growing human mesenchymal stem cells from a cell population containing human mesenchymal stem cells;
    Rapidly proliferating human mesenchymal system in which the existence ratio of cells expressing Ror2 or Fzd5 in the separated, sorted and cultured cell populations is quantified to determine whether each cell population is acceptable or not, and only successful cell populations are selected. A cell population of stem cells.
  15. ヒト間葉系幹細胞が含まれる細胞集団から、抗LNGFRモノクロナール抗体および抗Thy1モノクロナール抗体で染色した細胞をFCMで解析し、LNGFR Thy1共陽性細胞をセルソーティングし、培養プレートのウェルに同共陽性の各細胞を播種し、培養によりコンフルエントとなったウェルの細胞を分離及び選別した後、前記定量を行うことにより得られた請求項14記載の増殖の早いヒト間葉系幹細胞の細胞集団。 Cells stained with anti-LNGFR monoclonal antibody and anti-Thy1 monoclonal antibody from a cell population containing human mesenchymal stem cells are analyzed by FCM, LNGFR Thy1 co-positive cells are cell-sorted, and the cells are cultured in the wells of the culture plate. 15. The cell population of rapidly proliferating human mesenchymal stem cells according to claim 14, obtained by seeding each positive cell, separating and sorting cells in a well that became confluent by culture, and then performing the quantification.
  16. ヒト間葉系幹細胞が含まれる細胞集団から、抗Ror2モノクロナール抗体で染色した細胞をFCMで解析し、Ror2陽性細胞をセルソーティングし、培養プレートのウェルに同陽性の各細胞を播種し、培養によりコンフルエントとなったウェルの細胞を分離及び選別した後、前記定量を行うことにより得られた請求項14記載の増殖の早いヒト間葉系幹細胞の細胞集団。 From a cell population containing human mesenchymal stem cells, cells stained with anti-Ror2 monoclonal antibody are analyzed by FCM, Ror2-positive cells are cell-sorted, and each positive cell is seeded in a well of a culture plate and cultured. 15. The cell population of rapidly proliferating human mesenchymal stem cells according to claim 14, obtained by separating and sorting cells in a well that became confluent by the above, and then performing the quantification.
  17. 抗Ror2モノクローナル抗体である増殖の早いヒト間葉系幹細胞を特異的に認識するモノクローナル抗体。 A monoclonal antibody that specifically recognizes fast-growing human mesenchymal stem cells, which is an anti-Ror2 monoclonal antibody.
  18. クローン名が7C9である請求項17記載の増殖の早いヒト間葉系幹細胞を特異的に認識するモノクローナル抗体。 The monoclonal antibody that specifically recognizes fast-growing human mesenchymal stem cells according to claim 17, wherein the clone name is 7C9.
  19. 抗Fzd5モノクローナル抗体である増殖の早いヒト間葉系幹細胞を特異的に認識するモノクローナル抗体。 A monoclonal antibody that specifically recognizes fast-growing human mesenchymal stem cells, which is an anti-Fzd5 monoclonal antibody.
  20. クローン名が6F5である請求項19記載の増殖の早いヒト間葉系幹細胞を特異的に認識するモノクローナル抗体。 The monoclonal antibody specifically recognizing fast-growing human mesenchymal stem cells according to claim 19, whose clone name is 6F5.
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