US20170204374A1 - 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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US20170204374A1
US20170204374A1 US15/321,679 US201515321679A US2017204374A1 US 20170204374 A1 US20170204374 A1 US 20170204374A1 US 201515321679 A US201515321679 A US 201515321679A US 2017204374 A1 US2017204374 A1 US 2017204374A1
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cells
mesenchymal stem
human mesenchymal
stem cells
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Yumi IYOKU
Hideyuki Okano
Yo Mabuchi
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Purec Co Ltd
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    • C—CHEMISTRY; METALLURGY
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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
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic 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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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10001—Receptor protein-tyrosine kinase (2.7.10.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • the present invention relates to a method for quality evaluation of human mesenchymal stem cells, a method for isolation, selection and culture of human mesenchymal stem cells, a cell population of rapidly proliferating human mesenchymal stem cells, as well as monoclonal antibodies that specifically recognize rapidly proliferating human mesenchymal stem cells.
  • Mesenchymal stem cells are a kind of somatic stem cells which are increasingly used for clinical applications, after hematopoietic stem cells, because they have fewer ethical problems associated with cell collection and have differentiation potency into various types of tissues such as bone, cartilage, fat, etc.
  • Mesenchymal stem cells can be isolated through relatively simple manipulations as described later, and therefore are widely used as materials for biomaterials, for example, by being induced to differentiate into cartilage, bone and others mainly in test tubes and then used for local transplantation.
  • Non-patent Document 1 As a method for isolation and culture of human mesenchymal stem cells, the culture method reported in Non-patent Document 1 is commonly used. However, a cell population obtained by such a conventional method contains many contaminant cells of less quality (which have lost their differentiation, proliferation and migration potency), and these contaminant cells serve as a factor causing further loss of quality because they affect the cells which should have inherently had potential.
  • Non-patent Documents 2 and 3 and Patent Document 1 antibodies against CD271 (LNGFR) and CD90 (Thy1) are used to select LNGFR + Thy1 + cells from human bone marrow, placental chorion, fat tissue, peripheral blood, dental pulp and so on, whereby human mesenchymal stem cells can be enriched.
  • LNGFR CD271
  • Thy1 + cells from human bone marrow, placental chorion, fat tissue, peripheral blood, dental pulp and so on, whereby human mesenchymal stem cells can be enriched.
  • the selected LINGFR + Thy1 + cells are subjected to single cell (clone) culture to select a rapidly expanding lot (REC: Rapidly Expanding Clone), whereby human mesenchymal stem cells excellent in proliferation potency, differentiation potency and migration potency can be obtained in high purity.
  • REC Rapidly Expanding Clone
  • the high purity human mesenchymal stem cells (RECs) thus obtained were found to have proliferation potency, differentiation potency and migration potency which were all 1000-fold or more higher than those of mesenchymal stem cells obtained by conventional methods.
  • single cell culture allows the formation of conditions free from contaminant cells and thus enables expansion culture while maintaining cell quality.
  • the resulting cells can be administered via the intravenous route, and therefore can be expected for use in serious systemic diseases such as bone and cartilage hypoplasia.
  • the present invention aims to provide a method for quality evaluation of human mesenchymal stem cells, a method for isolation, selection and culture of human mesenchymal stem cells, a cell population of rapidly proliferating human mesenchymal stem cells, as well as monoclonal antibodies that specifically recognize rapidly proliferating human mesenchymal stem cells.
  • the method for quality evaluation of human mesenchymal stem cells is characterized in that from a cell population containing human mesenchymal stem cells, rapidly proliferating human mesenchymal stem cells are isolated, selected and cultured, and cells expressing Ror2 or Fzd5 (or the abundance ratio thereof) in the cell population thus isolated, selected and cultured are quantified to determine whether or not each cell population is acceptable.
  • Ror2 or Fzd5 when expressed alone allows determination of whether or not a cell population is composed of RECs, and the determination can be made in a simpler manner because cultured cells may also be used for this purpose.
  • LNGFR is not expressed in cultured cells even when they are RECs, and Thy1 when expressed alone does not allow determination of whether or not a cell population is composed of RECs.
  • cells expressing Ror2 or Fzd5 may be quantified by using anti-Ror2 monoclonal antibody or anti-Fzd5 monoclonal antibody.
  • mRNA expression of Ror2 may be quantified by quantitative PCR, or alternatively, cells expressing Ror2 or Fzd5 may be quantified by immunostaining.
  • FCM flow cytometry
  • the method for isolation, selection and culture of human mesenchymal stem cells is characterized in that from a cell population containing human mesenchymal stem cells, rapidly proliferating human mesenchymal stem cells are isolated, selected and cultured, and cells expressing Ror2 or Fzd5 (or the abundance ratio thereof) in the cell population thus isolated, selected and cultured are quantified to determine whether or not each cell population is acceptable, thus selecting only the cell population(s) determined to be acceptable.
  • cells expressing Ror2 or Fzd5 may be quantified by using anti-Ror2 monoclonal antibody or anti-Fzd5 monoclonal antibody.
  • cells expressing mRNA of Ror2 may be quantified by quantitative PCR, or alternatively, cells expressing Ror2 or Fzd5 may be quantified by immunostaining.
  • the above step where rapidly proliferating human mesenchymal stem cells are isolated, selected and cultured may comprise a step where the cell population containing human mesenchymal stem cells is analyzed by FCM for cells stained simultaneously with anti-LNGFR monoclonal antibody and anti-Thy1 monoclonal antibody to thereby effect cell sorting of LNGFR + Thy1+ cells.
  • the above step where rapidly proliferating human mesenchymal stem cells are isolated, selected and cultured may alternatively comprise a step where the cell population containing human mesenchymal stem cells is analyzed by FCM for cells stained with anti-Ror2 monoclonal antibody to thereby effect cell sorting of Ror2 + cells.
  • the above methods may each comprise a step where the above cell population is directly prepared from cells derived from each tissue, including bone marrow.
  • the method comprising the step of effecting cell sorting of Ror2 + cells may comprise a step where the above cell population is prepared by adherent culture of cells derived from each tissue, including bone marrow. This is because cultured cells of RECs are not positive for LNGFR but are positive for Ror2.
  • the above cell sorting step may comprise a step where the positive cells are seeded in wells of a culture plate and the cells in each well reaching confluence upon culture are then isolated and selected.
  • the cell population of rapidly proliferating human mesenchymal stem cells is characterized in that from a cell population containing human mesenchymal stem cells, rapidly proliferating human mesenchymal stem cells are isolated, selected and cultured, and cells expressing Ror2 or Fzd5 (or the abundance ratio thereof) in the cell population thus isolated, selected and cultured are quantified to determine whether or not each cell population is acceptable, thus selecting only the cell population(s) determined to be acceptable.
  • the cell population containing human mesenchymal stem cells may be analyzed by FCM for cells stained simultaneously with anti-LNGFR monoclonal antibody and anti-Thy1 monoclonal antibody to thereby effect cell sorting of LNGFR + Thy1+ cells, and these dual positive cells may be seeded in wells of a culture plate and the cells in each well reaching confluence upon culture may be isolated and selected, prior to the above quantification.
  • the cell population containing human mesenchymal stem cells may be analyzed by FCM for cells stained with anti-Ror2 monoclonal antibody to thereby effect cell sorting of Ror2 + cells, and these positive cells may be seeded in wells of a culture plate and the cells in each well reaching confluence upon culture may be isolated and selected, prior to the above quantification.
  • a novel monoclonal antibody according to the present invention is anti-Ror2 monoclonal antibody whose clone name is 7C9.
  • another novel monoclonal antibody according to the present invention is anti-Fzd5 monoclonal antibody whose clone name is 6F5.
  • Proteins encoded by the identified two genes are expressed specifically in RECs, and their expression is not observed in cell populations of less quality. Moreover, these genes are essential for maintaining the undifferentiated state of RECs, and they serve as effective indicators which are closely related to cell functions and ensure cell performance, but not serve as mere biomarkers, in light of the following results: 1) inhibition of their expression includes loss of cell performance; and 2) forced expression of these genes allows prolonged undifferentiated state, etc.
  • the present invention enables the provision of a method for quality evaluation of human mesenchymal stem cells, a method for isolation, selection and culture of human mesenchymal stem cells, a cell population of rapidly proliferating human mesenchymal stem cells, as well as monoclonal antibodies that specifically recognize rapidly proliferating human mesenchymal stem cells.
  • FIG. 1 shows the steps of selection, isolation, culture and quality evaluation of high quality mesenchymal stem cells (RECs).
  • FIG. 2 shows the results compared for various parameters among RECs, MECs and SECs.
  • FIG. 3 shows figures and graphs which indicate that Ror2 and Fzd5 are expressed specifically in RECs.
  • FIG. 4 shows figures and graphs which indicate that inhibition of Fzd5 induces loss of cell properties in RECs.
  • FIG. 5 shows photographs and graphs which indicate that forced expression of Fzd5 includes the undifferentiated nature of RECs.
  • FIG. 6 shows photographs and graphs which indicate the stainability of newly prepared anti-Fzd5 monoclonal antibody.
  • FIG. 7 shows photographs and figures which indicate the stainability of newly prepared anti-Ror2 monoclonal antibody.
  • FIG. 8 shows a schematic diagram for quality evaluation of cultured MSCs using REC-specific antibody.
  • FIG. 1 illustrates the step of REC isolation by the single clone culture method.
  • Mononuclear cells are prepared from human bone marrow (or fat or placental chorion) and these bone marrow mononuclear cells are stained with anti-LNGFR and anti-Thy1 (LNGFR + Thy1 + cells are referred to as LT cells).
  • FCM Flow cytometry
  • anti-CD106 monoclonal antibody may be added to effect clone sorting of LNGFR + Thy1 + cells that are also strongly positive for CD106.
  • FIG. 1 further shows the evaluation of cultured cells using REC markers (anti-Ror2 and anti-Fzd5).
  • RECs collected from the 96-well plate are transferred to culture dishes or culture flasks on a well-by-well basis, and cultured to reach confluence (expansion culture).
  • the cells grown in adherent state are collected from all lots, and an aliquot (about 1 to 3 ⁇ 10 3 cells) of each lot is sampled and single-stained with a monoclonal antibody against each REC marker (anti-Ror2 or anti-Fzd5).
  • REC marker-positive cells are analyzed by flow cytometry to determine the ratio of the REC marker-positive cells in the collected cells (mRNA expression of Ror2 may be quantified by quantitative PCR, or alternatively, this ratio may be determined manually under a microscope).
  • clone sorting of LT cells may be replaced with clone sorting of Ror2 + cells (P6 and P7 in Table 1).
  • LT cells or Ror2 + cells may be selected and seeded in groups of two or more per well of a 96-well culture plate (expressed as “Multiple” in Table 1; P5, P9 and P14). In this case, however, the purity is lower than that in clone sorting.
  • the term “confluence” or “confluent” refers to a state where 90% or more of the culture vessel surface is coated with cultured cells.
  • the term “semi-confluence” or “semi-confluent” refers to a state where 70% to 80% of the culture vessel surface is coated with cultured cells.
  • the size and type of culture devices to be used may be changed as appropriate depending on the growth rate of cells.
  • the above cell population is directly prepared from cells derived from each tissue, including bone marrow.
  • the above cell population may be prepared by adherent culture of cells derived from each tissue, including bone marrow (P10 to P15 in Table 1; expressed as “Adherent cultured cells”).
  • bone marrow mononuclear cells are seeded on a medium supplemented with 10% to 20% serum and bFGF (at 37° C. under 1% to 5% CO 2 ) and cultured for about 2 weeks to collect fibroblast-like adherent cells (CFU-F) appearing after culture.
  • the step of preparing a cell population may comprise treatment of bone marrow with collagenase. Alternatively, this step may be designed such that a cell population is prepared from peripheral blood after G-CSF administration.
  • Step 2-2 the evaluation prior to shipment (Step 2-2) and Step 2-3)) is not always necessary, and adherent cultured cells may be used and subjected to FCM sorting to isolate Ror2 + cells, optionally followed by expansion culture, and the thus obtained cells may be provided for treatment in Step 2-5) and the subsequent steps prior to shipment, as in Process P15 in Table 1.
  • FIG. 2 shows the results obtained when RECs, MECs and SECs were compared for their cell performance using various parameter data.
  • FIG. 2A shows the results obtained when human bone marrow mononuclear cells were stained with antibodies against LNGFR and Thy1, followed by FCM analysis.
  • the area within the ellipse represents LT cells.
  • FIG. 2B is a schematic diagram for single cell isolation (clone sorting) of LT cells in a 96-well plate.
  • FIG. 2C is a graph showing the results measured for cell counts after single cell culture at fixed time intervals. RECs show a higher proliferation rate than MECs/SECs, and their count reaches 0.5 to 1 ⁇ 10 4 within about 2 weeks. A cell count of 0.5 to 1 ⁇ 10 4 is required to establish confluence in a well of a 96-well plate.
  • FIG. 2D shows the results obtained when RECs, MECs and SECs were induced to differentiate into bone and fat, followed by quantitative PCR to analyze gene expression specific to bone and fat cells. RECs were found to be particularly high in differentiation potency into fat cells when compared to MECs and SECs.
  • FIG. 2E is a graph showing the results obtained when RECs, MECs and SECs were seeded again in a 96-well plate by clone sorting and wells showing secondary colony formation were then counted and compared. Secondary colony formation serves as an indicator for self-replication potency which is indicative of the undifferentiated state. About 33% of RECs show secondary colony formation, whereas only a few colonies are formed from MECs/SECs.
  • FIG. 2F shows the results obtained when the following cell populations, each having been transformed with a Luc (luciferase) gene expression vector, i.e., WBMs (MSCs obtained in a standard manner; WBM is an abbreviation for Whole Bone Marrow), RECs and MECs/SECs, as well as non-luciferase-labeled WBMs prepared as a negative control group (Luc( ⁇ ) Cultured MSCs) were each administered to immunodeficient mice via the intravenous route, followed by intraperitoneal administration of luciferin serving as a substrate for Luc to observe luminescence from luciferase by using an in vitro detection system (IVIS) at 24 hours after transplantation.
  • Luc luciferase gene expression vector
  • the upper panel shows a graph obtained as follows: Luc luminescence intensity in each mouse was expressed numerically and, relative to the WBM MSC-transplanted group which was set to 100%, the luminescence ratio (%) was plotted for the mice transplanted with the other cells.
  • the lower panel shows images of Luc luminescence in the recipient mice of the respective groups.
  • the REC-transplanted mice show extremely low luminescence intensity in their lungs, thus indicating that RECs are rarely trapped into capillary vessels in the lungs, whereas MECs/SECs are trapped almost at the same level as WBMs (cultured MSCs obtained in a standard manner) and remain in the lungs.
  • RECs are a cell population excellent in proliferation potency, differentiation potency and migration potency, and are particularly advantageous in that they have migration potency comparable to that of MSCs in fresh bone marrow, in terms of being able to be administered systemically against intractable diseases as described later.
  • RECs are characterized by the following, when compared to normal MSCs:
  • RECs are the most undifferentiated cell population among human MSCs, and have the most similar properties to MSCs in bone marrow. Moreover, when compared to MECs/SECs or MSCs obtained in a standard manner, RECs are a fresh and less mutated cell population ensuring cell performance due to their higher differentiation, proliferation and migration potency.
  • FIG. 3A shows a comparison of Ror2 mRNA expression in RECs, MECs and SECs, as measured by quantitative PCR.
  • FIG. 3B shows a comparison of Fzd5 mRNA expression in RECs, MECs and SECs, as measured by quantitative PCR.
  • FIG. 3C shows a comparison of Fzd5 protein expression, as measured by Western blotting.
  • FIG. 3D shows photographs compared for intracellular localization of Fzd5 protein, as observed by immunofluorescent staining.
  • the expression of Fzd5 and Ror2 is specific to RECs.
  • the respective expression of Fzd5 and Ror2 when detected and quantified, would be effective as an indicator for cell quality evaluation of RECs.
  • the newly prepared anti-Fzd5 monoclonal antibody and anti-Ror2 monoclonal antibody are able to detect and quantify their target protein antigens in any technique selected from flow cytometry, Western blotting and immunofluorescent staining.
  • the RNA interference method is a technique to examine the function of a target gene by introduction of short RNA (shRNA) having a sequence complementary to target mRNA into cells to thereby disrupt the target mRNA.
  • FIG. 4 shows the results obtained from a series of experiments on cell properties in RECs when Fzd5 mRNA was disrupted by shRNA having a sequence complementary to Fzd5 (shFZD5), in comparison with the control group (shCTRL; shRNA having a random sequence not complementary to Fzd5).
  • FIG. 4A is a graph showing the mRNA level of Fzd5, as quantified by quantitative PCR, after introduction of shFZD5 or shCTRL into RECs.
  • shCTRL short hairpin control
  • FIG. 4B is a graph whose vertical axis plots cell counts in the group forced to express shFZD5 after introduction of shFZD5 or shCTRL into RECs, relative to cell counts in the control group which are set to 1, and whose horizontal axis plots the number of days after shRNA introduction.
  • RECs forced to express shFZD5 showed a sudden reduction in their cell count when compared to the control group, thus suggesting that Rzd5 inhibition would induce a reduction in proliferation potency.
  • FIG. 4C shows images of fat droplets stained with Oil-Red-O at 14 days of culture, after introduction of shFZD5 or shCTRL into RECs and the subsequent induction of their differentiation into fat cells. Fzd5 inhibition was found to induce a reduction in differentiation potency into fat cells, when compared to the control group.
  • FIG. 4D shows images stained by x-gal staining after introduction of shFZD5 or shCTRL into RECs, along with a graph which plots the frequency of cells having SA- ⁇ -gal activity in each cell population.
  • FIG. 4E is a graph showing the mRNA level of p16(INK4a) serving as an indicator for cellular aging, as quantified by quantitative PCR.
  • the control group was set to 100, the mRNA level of p16 in shFZD5-receiving RECs was about 300, thus indicating that inhibition of Fzd5 expression induced cellular aging.
  • FIG. 4F shows images observed for stress fiber formation by intracellular staining with anti-F-actin antibody in each cell population after introduction of shFZD5 or shCTRL into RECs, along with a graph which plots the averaged area (cell size) of individual cells in these respective cell populations.
  • the full-length cDNA of Fzd5 was forced to be expressed in RECs to cause constitutive expression of Fzd5 mRNA, and the effect thereof on cell functions was confirmed.
  • an expression vector is constructed to carry Fzd5 cDNA and a fluorescent protein GFP (Green Fluorescent Protein) in tandem to thereby allow co-expression of GFP in Fzd5 gene-receiving cells, so that the expression of the introduced gene can be confirmed under a fluorescence microscope.
  • GFP Green Fluorescent Protein
  • FIG. 5A shows photographs observed for morphology of GFP-expressing cells under a fluorescence microscope. These images show cell morphology at 28 days after gene transfer in the cell population transformed with Fzd5 cDNA and GFP (Fzd5) as well as the control group transformed with the GFP gene alone (CTRL).
  • FIG. 5B is a graph whose vertical axis plots cell counts of Fzd5-expressing RECs, relative to cell counts in the control group which are set to 1, and whose horizontal axis plots the number of days after gene transfer. RECs forced to express Fzd5 were found to retain their proliferation potency for a long period of time, when compared to the control group.
  • Fzd5-mediated stimulation of Wnt signaling would be expected to allow long-term culture amplification in a state maintaining the undifferentiated nature.
  • the extracellular region of a human Fzd5 antigen was used as an immunogen to immunize host mice and hybridomas were then prepared in accordance with standard procedures, followed by screening with Ba/F3 cells engineered to express the Fzd5 gene, thereby obtaining a novel anti-Fzd5 monoclonal antibody (clone name: 6F5).
  • This antibody was used to confirm whether or not the Fzd5 protein was able to be detected by various techniques.
  • FIG. 6A shows a histogram whose horizontal axis plots the fluorescence intensity of PE.
  • the shaded histogram in the figure represents a negative control where an isotype control was added as a primary antibody, while the open histogram represents the PE fluorescence intensity in the sample stained with 6F5.
  • the range indicated with the horizontal bar in the figure represents the range of Fzd5 + cells, while the numerical value represents the positive rate (%).
  • FIG. 6B shows the results of Western blotting obtained when intracellular proteins were prepared from three different clones of RECs and the Fzd5 protein was detected using 6F5 as a primary antibody.
  • intracellular proteins were prepared from the monkey kidney-derived cell line COST.
  • FIG. 6C shows images obtained when REC cells were stained with 6F5-biotin as a primary antibody and then fluorescently labeled with streptavidin (SAV)-Alexa 555, followed by observation under a fluorescence microscope.
  • SAV streptavidin
  • the anti-Fzd5 antibody (6F5) was found to be available for use in all of flow cytometry, Western blotting and immunofluorescent staining.
  • a human Ror2 antigen was used as an immunogen to newly prepare an anti-Ror2 antibody (clone name: 7C9).
  • FIG. 7A shows the results obtained when RECs were stained with 7C9-biotin as a primary antibody and then labeled with SAV-PE, followed by flow cytometry to detect PE fluorescence.
  • a sample prepared by adding an isotype control antibody as a primary antibody is used as a negative control.
  • FIG. 7A is a two-dimensional dot plot whose vertical axis plots FITC fluorescence (RECs are all negative because they are not stained) and whose horizontal axis plots PE fluorescence.
  • FIG. 7B is an image obtained when RECs were immunostained with 7C9-biotin as a primary antibody and fluorescently labeled with streptavidin-Alexa 488, and then observed for expression of the Ror2 protein under a fluorescence microscope. Most of these RECs were confirmed to express the Ror2 protein.
  • FIG. 7C shows the results obtained when fresh bone marrow cells were triple stained with LNGFR-APC, Thy1-FITC and Ror2-PE (monoclonal antibodies against their respective antigens), followed by flow cytometry analysis.
  • the left panel shows a figure whose vertical axis plots LNGFR expression and whose horizontal axis plots Thy1 expression, and the boxed area represents a LNGFR + Thy1+ cell population containing human MSCs with high frequency.
  • the right figure shows the LNGFR Thy1+ cell population extracted alone.
  • the horizontal axis plots FSC serving as an indicator for cell size, while the vertical axis plots PE fluorescence from anti-Ror2-biotin antibody (7C9) labeled with SAV-PE.
  • the boxed area represents the Ror2-positive area determined on the basis of the negative control, and the numerical value represents the positive rate (%).
  • clone 7C9 92.3% of LNGFR + Thy1+ cells are Ror2-positive; and hence clone 7C9 can be used as a selection marker for MSCs to replace LNGFR Thy1.
  • the Ror2 protein can be detected and quantified by flow cytometry and immunofluorescent staining ( FIGS. 7A and 7B ).
  • the newly prepared anti-Ror2 can also be used as a marker for MSCs contained in bone marrow ( FIG. 7C ).
  • the frequency (% content) of positive cells is measured by flow cytometry.
  • the frequency (% content) of positive cells may be measured under a fluorescence microscope, instead.
  • the present invention enables the provision of a technique for efficient isolation and culture of human mesenchymal stem cells available for use in the treatment of systemic diseases, and also enables quality evaluation on which to determine whether or not the resulting cell population is suitable for transplantation and/or exerts efficacy.
  • candidates suitable for cell isolation can be provided as reagents for isolation of mesenchymal stem cells by being immobilized on magnetic nanoparticles.
  • fluorescent substance-labeled antibodies or cell staining reagents can be provided for practical use.
  • Mesenchymal stem cells are not only used as materials for biomaterials, as previously known, but also can be expected to have various applications by taking advantage of their pluripotency, as exemplified by administration to myasthenia gravis, chronic rheumatism and other diseases, as well as co-transplantation as supporting cells to provide a tissue scaffold (niche) for cell therapy required for treatment of severe diseases including spinal cord injury, heart and vascular failure, chronic liver failure and so on.
  • tissue scaffold niche
  • the use of RECs retaining their migration potency would be expected to provide a therapeutic effect never seen before when applied to metabolic diseases such as systemic bone and cartilage diseases including hypophosphatasia, for which no therapy has been found, as well as GVHD and all other diseases whose treatment requires administration via the intravenous route.

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