WO2010016492A1 - Method for identifying and isolating stem cell from dental or periodontal tissue and stem cell obtained by the method - Google Patents

Method for identifying and isolating stem cell from dental or periodontal tissue and stem cell obtained by the method Download PDF

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WO2010016492A1
WO2010016492A1 PCT/JP2009/063811 JP2009063811W WO2010016492A1 WO 2010016492 A1 WO2010016492 A1 WO 2010016492A1 JP 2009063811 W JP2009063811 W JP 2009063811W WO 2010016492 A1 WO2010016492 A1 WO 2010016492A1
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cells
ssea
tissue
periodontal
derived
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PCT/JP2009/063811
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French (fr)
Japanese (ja)
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紀章 川邉
隆 山城
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国立大学法人 岡山大学
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    • 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

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  • the present invention relates to a method for identifying and obtaining a stem cell. Specifically, the present invention relates to a method for identifying and obtaining periodontal ligament stem cells using SSEA-4 as a marker, and stem cells obtained by the method. The present invention further relates to the use of the stem cells.
  • Stem cells are cells that have the ability to differentiate into various body tissues and organs, and are very useful in developmental research and regenerative medicine. In order to acquire these stem cells, a reliable and simple identification method is required. As a method for identifying a stem cell so far, for example, STRO-1 has been reported as one of markers for identifying a bone marrow mesenchymal stem cell. Recently, teeth and periodontal tissue have attracted attention as a source of stem cells. The reason for this is that acquisition is easier than bone marrow stem cells and the like, and there are fewer ethical problems like ES cells.
  • Non-Patent Document 1 So far, only a method using STRO-1 antibody has been reported as a method for identifying stem cells from human teeth (see Non-Patent Document 1). However, STRO-1 expression is not always constant, and there is a report that expression is not observed. In addition, since the molecule of STRO-1 antigen is not yet elucidated, its expression instability has not been elucidated yet.
  • SSEA-4 has been used as a marker for stem cells derived from teeth and periodontal tissues
  • the problem to be solved by the present invention is to develop a method for accurately and simply identifying and isolating stem cells from teeth and periodontal tissues, and efficiently obtaining stem cells from teeth and periodontal tissues. It was to apply to.
  • the present inventors have conducted intensive research in view of the above problems, and can identify and isolate stem cells reliably and efficiently from teeth or periodontal tissue-derived cells using SSEA-4 as a marker.
  • SSEA-4 derived from periodontal tissue
  • stem cells expressing SSEA-4 derived from periodontal tissue also referred to as SSEA-4 positive stem cells
  • SSEA-4 positive stem cells have a high differentiation ability capable of differentiating into any tissue derived from ectoderm, mesoderm and endoderm.
  • the invention has been completed.
  • the present invention (1) Obtain cells from teeth or periodontal tissue, A method for identifying stem cells derived from teeth or periodontal tissue, wherein SSEA-4 positive cells are detected from the obtained cells; (2) The method according to (1), wherein the detection is performed using a flow cytometer; (3) Obtain cells from teeth or periodontal tissue, A method for obtaining stem cells from teeth or periodontal tissue, comprising isolating SSEA-4 positive cells from the obtained cells; (4) The method according to (1), wherein the isolation is performed using a flow cytometer or magnetic beads; (5) isolated SSEA-4 positive stem cells obtained from teeth or periodontal tissue; (6) Isolated SSEA-4-positive stem cells obtained by the method according to (3) or (4); (7) The stem cell according to (5) or (6), which is derived from the periodontal ligament; (8) The stem cell according to any one of (5) to (7), which can differentiate into any tissue derived from ectoderm, mesoderm and endoderm; (9) A method for producing an ec
  • stem cells can be reliably and easily identified and isolated from cells derived from teeth or periodontal tissues.
  • highly useful stem cells having high differentiation ability can be obtained.
  • FIG. 1 is a photograph of primary human periodontal ligament cells cultured in a plastic culture dish.
  • the upper left and lower left panels are cells after 24 hours of culture, and the upper right and lower right panels are cells after 7 days of culture. Scale bars are 400 ⁇ m (upper left and upper right panels), 100 ⁇ m (lower left and lower right panels).
  • FIG. 2A is a diagram showing the results of analyzing the expression of various antigens in cultured human periodontal ligament cells using flow cytometry.
  • the numerical value on the horizontal axis indicates relative fluorescence intensity, and is 0, 10 2 , 10 3 , 10 4 , 10 5 from the left, the numerical value on the vertical axis indicates the number of cells, and 0, 20, 40, 60, 80, 100.
  • FIG. 2B is a diagram showing the results of analyzing the expression of various antigens in cultured human periodontal ligament cells using flow cytometry.
  • B Cytokine receptor.
  • the numerical value on the horizontal axis indicates relative fluorescence intensity, and is 0, 10 2 , 10 3 , 10 4 , 10 5 from the left, the numerical value on the vertical axis indicates the number of cells, and 0, 20, 40, 60, 80, 100.
  • FIG. 2C is a diagram showing the results of analyzing the expression of various antigens in cultured human periodontal ligament cells using flow cytometry.
  • C Blood cell marker.
  • the numerical value on the horizontal axis indicates relative fluorescence intensity, and is 0, 10 2 , 10 3 , 10 4 , 10 5 from the left, the numerical value on the vertical axis indicates the number of cells, and 0, 20, 40, 60, 80, 100.
  • FIG. 2D is a diagram showing the results of analyzing the expression of various antigens in cultured human periodontal ligament cells using flow cytometry.
  • D Other.
  • the numerical value on the horizontal axis indicates relative fluorescence intensity, and is 0, 10 2 , 10 3 , 10 4 , 10 5 from the left, the numerical value on the vertical axis indicates the number of cells, and 0, 20, 40, 60, 80, 100.
  • 3A to 3E show the results of examining the expression of embryonic stem cell-related molecules in cultured human periodontal ligament cells.
  • FIG. 3A shows analysis of expression of ES cell-related molecules and detection of alkaline phosphatase activity by fluorescent immunostaining. From the top left, the expression of SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 is shown.
  • FIG. 3B shows the results of examining the expression of SSEA-1, SSEA-3, and SSEA-4 in cultured human periodontal ligament cells using flow cytometry.
  • FIG. 3C shows the results of examining changes in SSEA-1, SSEA-3, and SSEA-4 antigen expression when differentiation of cultured human periodontal ligament cells was induced using retinoic acid. “Cont” represents a retinoic acid non-added group, and “RA” represents a retinoic acid added group. The vertical axis is the percentage of positive cells.
  • 3D is a diagram showing a comparison of telomere lengths of SSEA-4 positive cells and negative cells in cultured human periodontal ligament cells.
  • the vertical axis represents the number of fluorescent units.
  • FIG. 3E is a diagram showing a comparison of the proliferation ability of SSEA-4 positive cells and negative cells in cultured human periodontal ligament cells.
  • the vertical axis represents absorbance.
  • 4A to I show the results of examining the differentiation potential of SSEA-4 positive stem cells derived from human periodontal ligament of the present invention into mesodermal tissues.
  • the control group (Cont) is cultured in a basic medium to which no differentiation-inducing factor is added.
  • 4A to 4D show the results of examining differentiation into adipocytes.
  • FIG. 4A shows the result of oil red O staining after culturing in an adipocyte differentiation induction medium for 21 days. The scale bar is 50 ⁇ m.
  • FIG. 4B shows the results of examining the expression of adipocyte markers using RT-PCR.
  • 4A, 4C and 4D show that Adipo was cultured in adipocyte differentiation induction medium.
  • FIG. 4C shows the result of quantitative analysis of lipid droplet formation by oil red O staining.
  • 4A, 4C and 4D show that Adipo was cultured in adipocyte differentiation induction medium.
  • FIG. 4D shows the analysis result by ELISA of adiponectin secretion in the cell culture supernatant.
  • FIG. 4D shows the adiponectin density
  • 4A, 4C and 4D show that Adipo was cultured in adipocyte differentiation induction medium.
  • 4E to 4G show the results of examining differentiation into osteoblasts.
  • FIG. 4E shows the results of alizarin red S staining after culturing in an osteoblast differentiation induction medium for 21 days. The scale bar is 50 ⁇ m.
  • FIG. 4F shows the results of examining the expression of osteoblast markers using RT-PCR.
  • FIGS. 4F and 4G show Osteo cultured in osteoblast differentiation medium.
  • FIG. 4G shows the quantitative analysis results of Ca deposited on the cells. The vertical axis of FIG.
  • FIG. 4G shows the amount of Ca deposition ( ⁇ g / cm 2 ) per unit area.
  • FIGS. 4F and 4G show Osteo cultured in osteoblast differentiation medium.
  • 4H to I show the results of examining differentiation into chondrocytes.
  • FIG. 4H shows the result of culturing in a chondrocyte differentiation-inducing medium for 21 days, embedding in paraffin, preparing a section, and staining with Alcian blue. The scale bar is 50 ⁇ m.
  • FIG. 4I shows the results of examining the expression of chondrocyte markers using RT-PCR. In FIG. I, D0 shows the 0th day of culture, and D21 shows the 21st day of culture. The vertical axis in FIG. 4C represents absorbance.
  • FIG. I shows the results of examining differentiation into chondrocytes.
  • FIG. 4H shows the result of culturing in a chondrocyte differentiation-inducing medium for 21 days, embedd
  • FIG. 5 shows the results of examining the differentiation ability of human periodontal ligament-derived SSEA-4-positive human periodontal ligament cells of the present invention into ectodermal tissue.
  • the control group Cont
  • FIG. 5A shows the results of fluorescence immunostaining of neuron-specific enolase (NSE) after culturing in a neuronal differentiation induction medium for 3 days.
  • the scale bar is 50 ⁇ m.
  • FIG. 5B shows the results of examining the expression of neural cell markers using RT-PCR. Neuro indicates that the cells were cultured in a neuronal differentiation induction medium.
  • FIG. 6A to 6D show the results of examining the differentiation ability of human periodontal ligament cells derived from human periodontal ligament cells of the present invention into endoderm tissues.
  • the control group Cont
  • FIG. 6A shows the results of fluorescent immunostaining of albumin after culturing in a hepatocyte differentiation-inducing medium for 21 days.
  • FIG. 6B shows the results of periodate-Schiff (PAS) staining. Arrowheads indicate binuclear cell nuclei. The scale bar is 50 ⁇ m.
  • FIG. 6C shows the results of examining the expression of hepatocyte markers using RT-PCR.
  • FIG. 6D shows the analysis result of albumin secretion in the cell culture supernatant by ELISA. Hepato indicates that the cells were cultured in a hepatocyte differentiation induction medium.
  • the present invention provides a stem cell derived from a tooth or periodontal tissue, wherein cells are obtained from the tooth or periodontal tissue, and SSEA-4 positive cells are detected from the obtained cells. It relates to a method for identifying.
  • the present invention provides a stem cell from a tooth or periodontal tissue, wherein cells are obtained from the tooth or periodontal tissue, and SSEA-4 positive cells are isolated from the obtained cells. On how to get.
  • SSEA-4 has a more stable and stable antigen expression than STRO-1, which has been used for identification of human dental stem cells. Therefore, stem cells derived from teeth or periodontal tissues can be obtained more accurately and efficiently. Can be identified or isolated.
  • SSEA-4 is used as a marker for stem cells derived from teeth and periodontal tissues, and there is no example in which the differentiation potential of SSEA-4 positive stem cells derived from teeth and periodontal tissues is examined.
  • the cell source in the above method of the present invention is a tooth or periodontal tissue, and for example, tissues such as periodontal ligament, dental pulp, cementum, alveolar bone, gingiva, and tooth germ can be the cell source.
  • Periodontal ligament is preferred as the cell source because it is relatively easy to obtain by dental treatment such as extraction.
  • a method for obtaining a periodontal ligament from a tooth is known.
  • a periodontal ligament can be collected from a washed tooth using a scalpel.
  • Other tissue acquisition means and methods are also known.
  • the cells can be obtained by digesting the obtained tissue.
  • Tissue digestion methods are also known, and for example, digestion can be performed in a solution containing a proteolytic enzyme.
  • SSEA-4 positive cells are detected and / or sorted and / or identified from the obtained cells.
  • various methods such as flow cytometry and magnetic bead method are known and can be applied to the present invention.
  • a preferred method is flow cytometry.
  • the cells Prior to subjecting the cells to flow cytometry, the cells are labeled or stained with a substance specific for SSEA-4.
  • An antibody staining method is exemplified as a method generally used for labeling or staining of cells.
  • the cells are stained with a combination of a primary antibody specific for the cell's SSEA-4 and a secondary antibody with a detectable fluorescent label and subjected to flow cytometry.
  • a flow cytometer is generally used.
  • the use of a flow cytometer is a preferable means and method for detecting and / or sorting and / or identifying the SSEA-4 positive cells of interest in the present invention.
  • SSEA-4 positive cells selected and identified as described above can be isolated using a known method. Examples of the cell isolation method include flow cytometry and magnetic bead method. If a flow cytometer is used, target SSEA-4-positive cells can be isolated more accurately.
  • the present invention relates to SSEA-4 positive stem cells obtained from teeth or periodontal tissue.
  • the periodontal tissue is the periodontal ligament.
  • the SSEA-4 positive cells obtained according to the present invention are isolated. Such cells can be obtained using SSEA-4 as a marker using the method of the present invention described above. Tooth or periodontal tissue-derived stem cells obtained according to the present invention have a high pluripotency capable of differentiating not only into the mesoderm but also into any tissue derived from the ectoderm / endoderm. It is characteristic to have.
  • the present invention provides a method for producing an ectoderm-derived tissue and / or mesoderm-derived tissue and / or endoderm-derived tissue, characterized in that SSEA-4 positive stem cells are differentiated.
  • SSEA-4-positive stem cells used in these methods are those obtained from the teeth or periodontal tissue according to the present invention.
  • Conditions for differentiating SSEA-4 positive stem cells into ectoderm-derived tissue, mesoderm-derived tissue or endoderm-derived tissue are known to those skilled in the art.
  • this invention also includes the structure
  • the SSEA-4 positive cells of the present invention can differentiate into any of ectoderm-derived tissue, mesoderm-derived tissue, and endoderm-derived tissue is very important in developmental research and other research fields as well as in the field of regenerative medicine. Useful for. For example, in a patient with missing teeth or periodontal tissue, SSEA-4 positive stem cells are isolated from the periodontal ligament after wisdom tooth extraction, and differentiated into teeth or periodontal tissue, so that the missing tooth or periodontal tissue It can also be played back. Therefore, the present invention isolates SSEA-4 positive stem cells from the periodontal ligament, differentiates them into teeth or periodontal tissue, and transplants the differentiated tissue to the affected area. Provide a playback method.
  • the present invention also provides the use of periodontal ligament-derived SSEA-4 positive cells for producing a tooth or periodontal tissue used for regeneration of a missing tooth or periodontal tissue. Furthermore, the present invention relates to a method for producing a tooth or periodontal tissue used for regeneration of a missing tooth or periodontal tissue, wherein periodontal ligament-derived SSEA-4 positive cells are transformed into the tooth or periodontal tissue in a differentiation medium. A method characterized by differentiating is provided. Conditions for differentiating SSEA-4 positive cells derived from the basement membrane into teeth or periodontal tissues in a differentiation medium are known to those skilled in the art and can be appropriately selected and used.
  • Example 1 Identification and isolation of stem cells from periodontal ligament and expression of various factors in the obtained stem cells
  • the patient's teeth were washed twice with Ca 2+ , Mg 2+ -free phosphate buffer (phosphate-buffered saline: PBS (-)), and then the root surface was shaved using a surgical blade.
  • Periodontal ligaments were collected so that there was no damage.
  • the periodontal ligaments obtained were modified with low glucose Dulbecco's modified Eagle medium (DMEM; Invitrogen, containing 5 mg / mL collagenase type II (Worthington, Lakewood, NJ, USA) and 2.5 mg / mL dispase I (Roche, Penzbeg, Germany). Carlsbad, CA, USA) at 37 ° C. for 60 minutes.
  • DMEM low glucose Dulbecco's modified Eagle medium
  • the resulting human periodontal ligament cells were treated with 15% heat inactivated fetal bovine serum (HIFBS; HyClone, Logan, UT, USA) and 1% penicillin / streptomycin (final concentrations of 100 U / mL and 100 ⁇ g / 100%, respectively). Incubated with low glucose DMEM containing mL; Invitrogen). Half of the medium was changed twice a week. Under the above conditions, human periodontal ligament cells cultured in a plastic culture dish were observed with a phase contrast microscope. The cells were spindle-shaped and had a mononuclear fibroblast-like morphology and were morphologically the same as bone marrow mesenchymal stem cells. After 24 hours of culture, symmetric cell division was observed (FIG. 1, upper left and lower left panels). After 7 days of culture, a mesh network was formed (upper right and lower right panels in FIG. 1).
  • the expression of various antigens in cultured human periodontal ligament cells was analyzed using flow cytometry.
  • the flow cytometry procedure was as follows.
  • the primary antibody was added to the cell suspension and allowed to react on ice for 60 minutes.
  • a fluorescently labeled secondary antibody was further added after the primary antibody reaction and allowed to react on ice for 30 minutes.
  • 7-amino-actinomycin D 7-AAD; BD Biosciences, San San Jose, CA, USA
  • Fig. 2 shows the results of flow cytometry.
  • Cultured human periodontal ligament cells exhibited the same antigen expression pattern as bone marrow mesenchymal stem cells, such as expressing CD29, CD44, CD71, CD73, CD90, and CD105.
  • Fluorescent immunocytochemistry Cells were fixed with 4% PFA for 10 minutes, then permeabilized and blocked for 30 minutes with PBS (-) containing 0.1% nonidet P-40 and 10% normal goat serum (Sigma) It was. The primary antibody was reacted at room temperature for 60 minutes, and the secondary antibody was reacted at room temperature for 30 minutes. Nuclear staining was performed using 4 ′, 6-diamidino-2-phenylindole (DAPI; Sigma). The stained cells were observed under a fluorescence microscope.
  • PBS -
  • DAPI 6-diamidino-2-phenylindole
  • Alkaline phosphatase activity was detected using the Alkaline Phosphatase Detection Kit (Chemicon, Temecula, CA, USA).
  • telomere length was measured using the Telomere PNA Kit / FITC for Flow Cytometry (Dako, Glostrup, Denmark).
  • the results are shown in FIG.
  • the cultured human periodontal ligament cells expressed a marker molecule of human embryonic stem cells (ES cells). From these results, it was found that undifferentiated human periodontal ligament stem cells can be identified using SSEA-4.
  • FIG. 3A The result of immunofluorescence staining is shown in FIG. 3A.
  • ES cell-related molecules surface antigen molecules: SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and transcription factors: OCT4, NANOG, SOX2 , REX1 expression was confirmed.
  • FIG. 3B shows the results of examining the expression of SSEA-1, SSEA-3, and SSEA-4 in cultured human periodontal ligament cells using flow cytometry. Many expression of SSEA-4 was observed.
  • FIG. 3D shows the results of comparison of the telomere length of SSEA-4 positive cells and negative cells in cultured human periodontal ligament cells. It was found that SSEA-4 positive cells had a long telomere length and SSEA-4 negative cells were short.
  • FIG. 3E shows the results of comparison of the proliferation ability of SSEA-4 positive cells and negative cells in cultured human periodontal ligament cells. SSEA-4 positive cells were highly proliferative and SSEA-4 negative cells were low.
  • Example 2 Differentiation ability of human periodontal ligament-derived SSEA-4-positive stem cells Differentiation ability of human periodontal ligament-derived SSEA-4 positive stem cells of the present invention into mesodermal, ectoderm, and endoderm tissues Investigated in vitro. The experimental procedure is shown below.
  • Single clone cells of SSEA-4 positive cells were isolated from cultured human periodontal ligament cells using flow cytometry, and the following differentiation induction was performed.
  • Induction of differentiation into adipocytes is 10% HIFBS, 1% penicillin / streptomycin, 1 ⁇ M dexamethasone (Sigma), 0.2 mM indomethacin (Sigma), 0.01 mg / mL insulin (Sigma), 0.5 mM 3-isobutyl-1 -Cultured in high glucose DMEM (Invitrogen) containing methylxanthine (Sigma) for 21 days.
  • the cultured cells were stained with oil red O (Sigma) and observed under an optical microscope. Secretion of adiponectin in the cell culture supernatant was measured using Human® Adiponectin / Acrp30® Immunoassay (R & D® Systems, Minneapolis, MN, USA).
  • Induction of differentiation into osteoblasts is minimal essential alpha medium ( ⁇ -MEM containing 10% HIFBS, 1% penicillin / streptomycin, 100 nM dexamethasone, 0.05 mM L-ascorbic acid (Sigma), 10 mM ⁇ -glycerophosphate (Sigma). Culturing was carried out for 21 days in Invitrogen). The cultured cells were stained with alizarin red S (Sigma) and observed under an optical microscope. Calcium deposited on the cells was measured using Calcium E-Test Wako (Wako Pure Chemical, Tokyo, Japan).
  • 3 ⁇ 10 5 cells were first dispensed into a 15 mL polypropylene conical tube (Corning, Corning, NY, USA) and centrifuged to form a cell mass. This cell mass was divided into 1% penicillin / streptomycin, 100 nM dexamethasone, 1 mM sodium pyruvate (Sigma), 0.17 mM L-ascorbic acid, 0.35 mM L-proline (Sigma), ITS + Universal Culture Supplement Premix (BD Biosciences), The cells were cultured for 21 days in high glucose DMEM containing 10 ng / mL TGF- ⁇ 3 (R & D Systems). The cultured cells were embedded in paraffin to prepare sections, stained with Alcian blue (Sigma), and observed under an optical microscope.
  • Induction of differentiation into nerve cells was performed by culturing for 24 hours in low glucose DMEM containing 15% HIFBS, 1% penicillin / streptomycin, 10 ng / mL bFGF (R & D Systems, polisMinneapolis, MN, USA), and then 2% dimethyl sulfoxide (DMSO).
  • DMEM dimethyl sulfoxide
  • the first stage is 2% HIFBS, 1% penicillin / streptomycin, 100 ng / mL aFGF (R & D Systems) -containing low glucose DMEM for 5 days
  • the second stage is 2% HIFBS, 1%
  • the third stage is 2% HIFBS, 1% penicillin / streptomycin, 20 ng / mL HGF, 10 nM dexamethasone, 1X insulin-transferrin -selenium-X (Invitrogen), 10 ng / mL Oncostatin M (R & D Systems) -containing low glucose DMEM was cultured for 11 days.
  • the cultured cells were stained with an anti-albumin antibody and an Alexa-Fluor-594 labeled secondary antibody and observed under a fluorescence microscope. Intracellular glycogen was detected using Periodic acid-schiff (PAS) holding kit (Sigma). Albumin secretion in the cell culture supernatant was measured using Human® serum® albumin assay (Cygnous Technologies, Southport, NC, USA).
  • PAS Periodic acid-schiff
  • FIGS. 5A to B show the results of examination of differentiation ability into ectoderm tissues.
  • 6A to 6D show the results of examining the differentiation ability into endoderm tissues. 63.6% (14/22) of SSEA-4 positive stem cell lines were shown to have pluripotency.
  • SSEA-4 positive stem cells were cultured in an adipocyte differentiation induction medium for 21 days, and then analyzed as follows.
  • FIG. 4A when oil red O staining was performed, a stained image was confirmed.
  • FIG. 4B the expression of adipocyte markers such as PPAR- ⁇ , ALBP, and LPL was confirmed by examining the expression of adipocyte markers by RT-PCR.
  • FIG. 4C when quantitative analysis of lipid droplet formation by oil red O staining was performed, about 5 times as much oil red O staining as the control group was observed.
  • FIG. 4D when adiponectin secretion was analyzed in the cell culture supernatant by ELISA, it was confirmed that the secretion increased with time.
  • SSEA-4 positive stem cells were cultured in an osteoblast differentiation induction medium for 21 days, and then analyzed as follows. As shown in FIG. 4E, when alizarin red S staining was performed, a stained image was confirmed. As shown in FIG. 4F, when the expression of osteoblast markers was examined by RT-PCR, the expression of osteoblast markers such as OPN and BSP was confirmed. As shown in FIG. 4G, when quantitative analysis of Ca deposited on the cells was performed, it was confirmed that the amount of deposited Ca increased with time.
  • SSEA-4 positive stem cells were cultured in a chondrocyte differentiation induction medium for 21 days, and then analyzed as follows. As shown in FIG. 4H, after the section was prepared by embedding in paraffin, stained with Alcian blue, a stained image was confirmed. As shown in FIG. 4I, when the expression of chondrocyte markers was examined by RT-PCR, the expression of chondrocyte markers such as COL II and COL X was confirmed.
  • SSEA-4 positive stem cells were cultured in a neuronal differentiation induction medium for 3 days, and then analyzed as follows. As shown in FIG. 5A, when immunofluorescent staining of neuron-specific enolase (NSE) was performed, a stained image was confirmed. As shown in FIG. 5B, the expression of neuronal markers such as NF-M and Tub- ⁇ 3 was confirmed by examining the expression of neuronal markers by RT-PCR.
  • NSE neuron-specific enolase
  • SSEA-4 positive stem cells were cultured in a hepatocyte differentiation-inducing medium for 21 days, and then analyzed as follows.
  • FIG. 6A when fluorescent immunostaining of albumin was performed, a stained image was confirmed.
  • FIG. 6B stained with periodic acid-Schiff (PAS), a stained image was confirmed.
  • FIG. 6C when the expression of hepatocyte markers was examined by RT-PCR, the expression of stem cell markers such as ALB and AFP was confirmed.
  • FIG. 6D when the albumin secretion in the cell culture supernatant was analyzed by ELISA, it was confirmed that the secretion increased with time.
  • the SSEA-4 positive stem cells of the present invention have a high differentiation ability capable of differentiating not only into mesodermal tissues but also into ectodermal tissues and endoderm tissues.
  • stem cells can be reliably and easily identified and isolated from teeth or periodontal tissue-derived cells, and the obtained stem cells can be any of ectoderm, mesoderm and endoderm. Since it has a high differentiation ability capable of differentiating into tissues, it is useful in developmental research and other research fields as well as in the field of regenerative medicine.

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Abstract

Provided is a method for identifying and isolating a stem cell originating from a dental or periodontal tissue by using SSEA-4 as a marker.  Also provided is as stem cell having a high differentiation ability which is obtained by the above-described method.

Description

歯または歯周組織からの幹細胞の同定・単離方法ならびに該方法により得られる幹細胞Method for identifying and isolating stem cells from teeth or periodontal tissue and stem cells obtained by the method
　本発明は、幹細胞の同定および取得方法に関する。詳細には、ＳＳＥＡ－４をマーカーとした歯根膜幹細胞の同定および取得方法、ならびに該方法により得られる幹細胞に関する。さらに本発明は、該幹細胞の使用にも関する。 The present invention relates to a method for identifying and obtaining a stem cell. Specifically, the present invention relates to a method for identifying and obtaining periodontal ligament stem cells using SSEA-4 as a marker, and stem cells obtained by the method. The present invention further relates to the use of the stem cells.
　幹細胞は、様々な身体の組織や臓器に分化する能力を有する細胞であり、発生の研究や再生医療において非常に有用である。これらの幹細胞を取得するには、確実でしかも簡便な同定方法が要求される。これまでに幹細胞を同定する方法としては、例えば、骨髄間葉系幹細胞を同定するマーカーの１つとしてＳＴＲＯ－１が報告されている。最近、歯および歯周組織が幹細胞のソースとして注目されている。その理由は、骨髄幹細胞などよりも取得が容易であること、ＥＳ細胞のような倫理的問題が少ないことなどが挙げられる。 Stem cells are cells that have the ability to differentiate into various body tissues and organs, and are very useful in developmental research and regenerative medicine. In order to acquire these stem cells, a reliable and simple identification method is required. As a method for identifying a stem cell so far, for example, STRO-1 has been reported as one of markers for identifying a bone marrow mesenchymal stem cell. Recently, teeth and periodontal tissue have attracted attention as a source of stem cells. The reason for this is that acquisition is easier than bone marrow stem cells and the like, and there are fewer ethical problems like ES cells.
　いままでのところ、ヒトの歯から幹細胞を同定する方法についてはＳＴＲＯ－１抗体を用いる方法のみが報告されている（非特許文献１参照）。しかし、ＳＴＲＯ－１の発現は必ずしも一定ではなく、発現が認められないという報告もある。また、ＳＴＲＯ－１抗原がどのような分子であるかについても未だに解明されていないので、その発現の不安定性についても未だに解明されていない。 So far, only a method using STRO-1 antibody has been reported as a method for identifying stem cells from human teeth (see Non-Patent Document 1). However, STRO-1 expression is not always constant, and there is a report that expression is not observed. In addition, since the molecule of STRO-1 antigen is not yet elucidated, its expression instability has not been elucidated yet.
　ＳＳＥＡ－４が、歯および歯周組織由来の幹細胞のマーカーとして用いられた例はなく、歯および歯周組織由来のＳＳＥＡ－４陽性幹細胞の分化能について調べた例もない。
Seo BM, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet, 2004 Jul 10-16; 364(9429): 149-55. There is no example in which SSEA-4 has been used as a marker for stem cells derived from teeth and periodontal tissues, and there has been no example of examining the differentiation potential of SSEA-4 positive stem cells derived from teeth and periodontal tissues.
Seo BM, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet, 2004 Jul 10-16; 364 (9429): 149-55.
　本発明の解決すべき課題は、歯や歯周組織から幹細胞を正確にしかも簡便に同定し、単離する方法を開発し、歯や歯周組織から幹細胞を効率よく得て、これを再生医療等に応用することであった。 The problem to be solved by the present invention is to develop a method for accurately and simply identifying and isolating stem cells from teeth and periodontal tissues, and efficiently obtaining stem cells from teeth and periodontal tissues. It was to apply to.
　本発明者らは、上記課題に鑑みて鋭意研究を行い、ＳＳＥＡ－４をマーカーとして用いて歯または歯周組織由来の細胞から確実かつ効率的に幹細胞を同定し、単離できること、そして、歯または歯周組織由来のＳＳＥＡ－４を発現する幹細胞（ＳＳＥＡ－４陽性幹細胞ともいう）は外胚葉、中胚葉および内胚葉由来のいずれの組織へも分化できる高い分化能を持つことを見出し、本発明を完成させるに至った。 The present inventors have conducted intensive research in view of the above problems, and can identify and isolate stem cells reliably and efficiently from teeth or periodontal tissue-derived cells using SSEA-4 as a marker. Alternatively, it has been found that stem cells expressing SSEA-4 derived from periodontal tissue (also referred to as SSEA-4 positive stem cells) have a high differentiation ability capable of differentiating into any tissue derived from ectoderm, mesoderm and endoderm. The invention has been completed.
　すなわち本発明は：
　（１）歯または歯周組織から細胞を得て、
　　　　得られた細胞のなかからＳＳＥＡ－４陽性細胞を検出する
ことを特徴とする、歯または歯周組織由来の幹細胞を同定する方法；
　（２）検出がフローサイトメーターを用いて行われる（１）記載の方法；
　（３）歯または歯周組織から細胞を得て、
　　　　得られた細胞のなかからＳＳＥＡ－４陽性細胞を単離する
ことを特徴とする、歯または歯周組織から幹細胞を得る方法；
　（４）単離がフローサイトメーターまたは磁気ビーズを用いて行われる（１）記載の方法；
　（５）歯または歯周組織から得られる、単離されたＳＳＥＡ－４陽性幹細胞；
　（６）（３）または（４）記載の方法により得られる単離されたＳＳＥＡ－４陽性幹細胞；
　（７）歯根膜由来である（５）または（６）記載の幹細胞；
　（８）外胚葉、中胚葉および内胚葉由来のいずれの組織へも分化できる（５）～（７）のいずれかに記載の幹細胞；
　（９）（５）～（７）のいずれかに記載の幹細胞を分化させることを特徴とする、外胚葉由来組織の製造方法；
　（１０）（５）～（７）のいずれかに記載の幹細胞を分化させることを特徴とする、中胚葉由来組織の製造方法；
　（１１）（５）～（７）のいずれかに記載の幹細胞を分化させることを特徴とする、内胚葉由来組織の製造方法；
　（１２）（９）～（１１）のいずれかに記載の方法により得られる組織；
　（１３）歯根膜からＳＳＥＡ－４陽性幹細胞を単離し、これを歯あるいは歯周組織に分化させ、分化組織を患部に移植することを特徴とする、欠損した歯あるいは歯周組織の再生方法；
　（１４）欠損した歯あるいは歯周組織の再生に用いられる歯あるいは歯周組織を製造するための、歯根膜由来のＳＳＥＡ－４陽性細胞の使用；ならびに
　（１５）欠損した歯あるいは歯周組織の再生に用いられる歯あるいは歯周組織の製造方法であって、歯根膜由来のＳＳＥＡ－４陽性細胞を分化培地にて歯あるいは歯周組織へと分化させることを特徴とする方法
を提供するものである。 That is, the present invention:
(1) Obtain cells from teeth or periodontal tissue,
A method for identifying stem cells derived from teeth or periodontal tissue, wherein SSEA-4 positive cells are detected from the obtained cells;
(2) The method according to (1), wherein the detection is performed using a flow cytometer;
(3) Obtain cells from teeth or periodontal tissue,
A method for obtaining stem cells from teeth or periodontal tissue, comprising isolating SSEA-4 positive cells from the obtained cells;
(4) The method according to (1), wherein the isolation is performed using a flow cytometer or magnetic beads;
(5) isolated SSEA-4 positive stem cells obtained from teeth or periodontal tissue;
(6) Isolated SSEA-4-positive stem cells obtained by the method according to (3) or (4);
(7) The stem cell according to (5) or (6), which is derived from the periodontal ligament;
(8) The stem cell according to any one of (5) to (7), which can differentiate into any tissue derived from ectoderm, mesoderm and endoderm;
(9) A method for producing an ectoderm-derived tissue, characterized in that the stem cell according to any one of (5) to (7) is differentiated;
(10) A method for producing a mesoderm-derived tissue, characterized in that the stem cell according to any one of (5) to (7) is differentiated;
(11) A method for producing an endoderm-derived tissue, characterized in that the stem cell according to any one of (5) to (7) is differentiated;
(12) A tissue obtained by the method according to any one of (9) to (11);
(13) A method for regenerating a missing tooth or periodontal tissue, comprising isolating SSEA-4 positive stem cells from the periodontal ligament, differentiating the stem cells into teeth or periodontal tissue, and transplanting the differentiated tissue to the affected area;
(14) Use of periodontal ligament-derived SSEA-4 positive cells to produce a tooth or periodontal tissue used for regeneration of the missing tooth or periodontal tissue; and (15) of the missing tooth or periodontal tissue. A method for producing a tooth or periodontal tissue used for regeneration, comprising the step of differentiating periodontal ligament-derived SSEA-4 positive cells into a tooth or periodontal tissue in a differentiation medium. is there.
　本発明によれば、歯または歯周組織由来の細胞から確実、簡便かつ効率的に幹細胞を同定し、単離することができる。また、本発明により、高い分化能を持つ有用性の高い幹細胞が得られる。 According to the present invention, stem cells can be reliably and easily identified and isolated from cells derived from teeth or periodontal tissues. In addition, according to the present invention, highly useful stem cells having high differentiation ability can be obtained.
図１は、プラスチック培養皿にて培養した初代ヒト歯根膜細胞の写真である。左上および左下パネルは培養２４時間後、右上および右下パネルは培養７日後の細胞である。スケールバーは４００μｍ（左上および右上パネル）、１００μｍ（左下および右下パネル）。FIG. 1 is a photograph of primary human periodontal ligament cells cultured in a plastic culture dish. The upper left and lower left panels are cells after 24 hours of culture, and the upper right and lower right panels are cells after 7 days of culture. Scale bars are 400 μm (upper left and upper right panels), 100 μm (lower left and lower right panels). 図２Ａは、フローサイトメトリーを用いて培養ヒト歯根膜細胞における各種抗原の発現を解析した結果を示す図である。Ａ：接着分子。図中、横軸の数値は相対的な蛍光強度を示し、左から０、１０２、１０３、１０４、１０５であり、縦軸の数値は細胞数を示し、下から０、２０、４０、６０、８０、１００である。FIG. 2A is a diagram showing the results of analyzing the expression of various antigens in cultured human periodontal ligament cells using flow cytometry. A: Adhesion molecule. In the figure, the numerical value on the horizontal axis indicates relative fluorescence intensity, and is 0, 10 2 , 10 3 , 10 4 , 10 5 from the left, the numerical value on the vertical axis indicates the number of cells, and 0, 20, 40, 60, 80, 100. 図２Ｂは、フローサイトメトリーを用いて培養ヒト歯根膜細胞における各種抗原の発現を解析した結果を示す図である。Ｂ：サイトカイン受容体。図中、横軸の数値は相対的な蛍光強度を示し、左から０、１０２、１０３、１０４、１０５であり、縦軸の数値は細胞数を示し、下から０、２０、４０、６０、８０、１００である。FIG. 2B is a diagram showing the results of analyzing the expression of various antigens in cultured human periodontal ligament cells using flow cytometry. B: Cytokine receptor. In the figure, the numerical value on the horizontal axis indicates relative fluorescence intensity, and is 0, 10 2 , 10 3 , 10 4 , 10 5 from the left, the numerical value on the vertical axis indicates the number of cells, and 0, 20, 40, 60, 80, 100. 図２Ｃは、フローサイトメトリーを用いて培養ヒト歯根膜細胞における各種抗原の発現を解析した結果を示す図である。Ｃ：血球マーカー。図中、横軸の数値は相対的な蛍光強度を示し、左から０、１０２、１０３、１０４、１０５であり、縦軸の数値は細胞数を示し、下から０、２０、４０、６０、８０、１００である。FIG. 2C is a diagram showing the results of analyzing the expression of various antigens in cultured human periodontal ligament cells using flow cytometry. C: Blood cell marker. In the figure, the numerical value on the horizontal axis indicates relative fluorescence intensity, and is 0, 10 2 , 10 3 , 10 4 , 10 5 from the left, the numerical value on the vertical axis indicates the number of cells, and 0, 20, 40, 60, 80, 100. 図２Ｄは、フローサイトメトリーを用いて培養ヒト歯根膜細胞における各種抗原の発現を解析した結果を示す図である。Ｄ：その他。図中、横軸の数値は相対的な蛍光強度を示し、左から０、１０２、１０３、１０４、１０５であり、縦軸の数値は細胞数を示し、下から０、２０、４０、６０、８０、１００である。FIG. 2D is a diagram showing the results of analyzing the expression of various antigens in cultured human periodontal ligament cells using flow cytometry. D: Other. In the figure, the numerical value on the horizontal axis indicates relative fluorescence intensity, and is 0, 10 2 , 10 3 , 10 4 , 10 5 from the left, the numerical value on the vertical axis indicates the number of cells, and 0, 20, 40, 60, 80, 100. 図３Ａ～Ｅは、培養ヒト歯根膜細胞における胚性幹細胞関連分子の発現について調べた結果を示す。図３Ａは、蛍光免疫染色によるＥＳ細胞関連分子の発現の解析およびアルカリホスファターゼ活性の検出を示す。上段左から、ＳＳＥＡ－１、ＳＳＥＡ－３、ＳＳＥＡ－４、ＴＲＡ－１－６０、ＴＲＡ－１－８１の発現を示す。下段左から、ＯＣＴ４、ＮＡＮＯＧ、ＳＯＸ２、ＲＥＸ１、アルカリホスファターゼ活性の発現を示す。スケールバーは５０μｍ（蛍光像）および１００μｍ（ＡＬＰ）。3A to 3E show the results of examining the expression of embryonic stem cell-related molecules in cultured human periodontal ligament cells. FIG. 3A shows analysis of expression of ES cell-related molecules and detection of alkaline phosphatase activity by fluorescent immunostaining. From the top left, the expression of SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 is shown. From the lower left, the expression of OCT4, NANOG, SOX2, REX1, and alkaline phosphatase activities is shown. Scale bars are 50 μm (fluorescence image) and 100 μm (ALP). 図３Ｂは、フローサイトメトリーを用いて培養ヒト歯根膜細胞におけるＳＳＥＡ－１、ＳＳＥＡ－３、ＳＳＥＡ－４の発現を調べた結果を示す。FIG. 3B shows the results of examining the expression of SSEA-1, SSEA-3, and SSEA-4 in cultured human periodontal ligament cells using flow cytometry. 図３Ｃは、レチノイン酸を用いて、培養ヒト歯根膜細胞を分化誘導した時の、ＳＳＥＡ－１、ＳＳＥＡ－３、ＳＳＥＡ－４抗原発現の変化を調べた結果を示す。Ｃｏｎｔはレチノイン酸非添加群、ＲＡはレチノイン酸添加群であることを示す。縦軸は陽性細胞のパーセンテージである。FIG. 3C shows the results of examining changes in SSEA-1, SSEA-3, and SSEA-4 antigen expression when differentiation of cultured human periodontal ligament cells was induced using retinoic acid. “Cont” represents a retinoic acid non-added group, and “RA” represents a retinoic acid added group. The vertical axis is the percentage of positive cells. 図３Ｄは、培養ヒト歯根膜細胞におけるＳＳＥＡ－４陽性細胞と陰性細胞のテロメア長の比較を示す図である。縦軸は蛍光ユニット数を示す。FIG. 3D is a diagram showing a comparison of telomere lengths of SSEA-4 positive cells and negative cells in cultured human periodontal ligament cells. The vertical axis represents the number of fluorescent units. 図３Ｅは、培養ヒト歯根膜細胞におけるＳＳＥＡ－４陽性細胞と陰性細胞の増殖能の比較を示す図である。縦軸は吸光度を示す。FIG. 3E is a diagram showing a comparison of the proliferation ability of SSEA-4 positive cells and negative cells in cultured human periodontal ligament cells. The vertical axis represents absorbance. 図４Ａ～Ｉは、本発明のヒト歯根膜由来のＳＳＥＡ－４陽性幹細胞の中胚葉系組織への分化能を検討した結果を示す。図中、対照群（Ｃｏｎｔ）は分化誘導因子非添加の基本培地にて培養したものである。図４Ａ～Ｄは脂肪細胞への分化を調べた結果を示す。図４Ａは脂肪細胞分化誘導培地にて２１日間培養した後、オイルレッドＯ染色を行った結果を示す。スケールバーは５０μｍである。4A to I show the results of examining the differentiation potential of SSEA-4 positive stem cells derived from human periodontal ligament of the present invention into mesodermal tissues. In the figure, the control group (Cont) is cultured in a basic medium to which no differentiation-inducing factor is added. 4A to 4D show the results of examining differentiation into adipocytes. FIG. 4A shows the result of oil red O staining after culturing in an adipocyte differentiation induction medium for 21 days. The scale bar is 50 μm. 図４ＢはＲＴ－ＰＣＲを用いて脂肪細胞マーカーの発現を調べた結果を示す。図４Ｂ、４Ｃおよび４ＤのＡｄｉｐｏは脂肪細胞分化誘導培地で培養したことを示す。FIG. 4B shows the results of examining the expression of adipocyte markers using RT-PCR. 4A, 4C and 4D show that Adipo was cultured in adipocyte differentiation induction medium. 図４ＣはオイルレッドＯ染色による脂肪滴形成の定量的解析結果を示す。図４Ｂ、４Ｃおよび４ＤのＡｄｉｐｏは脂肪細胞分化誘導培地で培養したことを示す。FIG. 4C shows the result of quantitative analysis of lipid droplet formation by oil red O staining. 4A, 4C and 4D show that Adipo was cultured in adipocyte differentiation induction medium. 図４Ｄは細胞培養上清におけるアジポネクチン分泌のＥＬＩＳＡによる解析結果を示す。図４Ｄの縦軸は細胞培養上清中のアジポネクチン濃度（ｎｇ／ｍＬ）を示す。図４Ｂ、４Ｃおよび４ＤのＡｄｉｐｏは脂肪細胞分化誘導培地で培養したことを示す。FIG. 4D shows the analysis result by ELISA of adiponectin secretion in the cell culture supernatant. The vertical axis | shaft of FIG. 4D shows the adiponectin density | concentration (ng / mL) in a cell culture supernatant. 4A, 4C and 4D show that Adipo was cultured in adipocyte differentiation induction medium. 図４Ｅ～Ｇは骨芽細胞への分化を調べた結果を示す。図４Ｅは骨芽細胞分化誘導培地にて２１日間培養した後、アリザリンレッドＳ染色の結果を示す。スケールバーは５０μｍである。4E to 4G show the results of examining differentiation into osteoblasts. FIG. 4E shows the results of alizarin red S staining after culturing in an osteoblast differentiation induction medium for 21 days. The scale bar is 50 μm. 図４ＦはＲＴ－ＰＣＲを用いて骨芽細胞マーカーの発現を調べた結果を示す。図４Ｆおよび４ＧのＯｓｔｅｏは骨芽細胞分化誘導培地で培養したことを示す。FIG. 4F shows the results of examining the expression of osteoblast markers using RT-PCR. FIGS. 4F and 4G show Osteo cultured in osteoblast differentiation medium. 図４Ｇは細胞に沈着したＣａの定量的解析結果を示す。図４Ｇの縦軸は単位面積当りのＣａ沈着量（μｇ／ｃｍ２）を示す。図４Ｆおよび４ＧのＯｓｔｅｏは骨芽細胞分化誘導培地で培養したことを示す。FIG. 4G shows the quantitative analysis results of Ca deposited on the cells. The vertical axis of FIG. 4G shows the amount of Ca deposition (μg / cm 2 ) per unit area. FIGS. 4F and 4G show Osteo cultured in osteoblast differentiation medium. 図４Ｈ～Ｉは軟骨細胞への分化を調べた結果を示す。図４Ｈは軟骨細胞分化誘導培地にて２１日間培養した後、パラフィン包埋して切片作成後、アルシアンブルー染色した結果を示す。スケールバーは５０μｍである。4H to I show the results of examining differentiation into chondrocytes. FIG. 4H shows the result of culturing in a chondrocyte differentiation-inducing medium for 21 days, embedding in paraffin, preparing a section, and staining with Alcian blue. The scale bar is 50 μm. 図４ＩはＲＴ－ＰＣＲを用いて軟骨細胞マーカーの発現を調べた結果を示す。図ＩのＤ０は培養０日目、Ｄ２１は培養２１日目を示す。図４Ｃの縦軸は吸光度を示す。FIG. 4I shows the results of examining the expression of chondrocyte markers using RT-PCR. In FIG. I, D0 shows the 0th day of culture, and D21 shows the 21st day of culture. The vertical axis in FIG. 4C represents absorbance. 図５は、本発明のヒト歯根膜由来のＳＳＥＡ－４陽性ヒト歯根膜細胞の外胚葉系組織への分化能を検討した結果を示す。図中、対照群（Ｃｏｎｔ）は分化誘導因子非添加の基本培地にて培養したものである。図５Ａは神経細胞分化誘導培地にて３日間培養した後、ニューロン特異的エノラーゼ（ＮＳＥ）の蛍光免疫染色を行った結果を示す。スケールバーは５０μｍである。図５ＢはＲＴ－ＰＣＲを用いて神経細胞マーカーの発現を調べた結果を示す。Ｎｅｕｒｏは神経細胞分化誘導培地で培養したことを示す。FIG. 5 shows the results of examining the differentiation ability of human periodontal ligament-derived SSEA-4-positive human periodontal ligament cells of the present invention into ectodermal tissue. In the figure, the control group (Cont) is cultured in a basic medium to which no differentiation-inducing factor is added. FIG. 5A shows the results of fluorescence immunostaining of neuron-specific enolase (NSE) after culturing in a neuronal differentiation induction medium for 3 days. The scale bar is 50 μm. FIG. 5B shows the results of examining the expression of neural cell markers using RT-PCR. Neuro indicates that the cells were cultured in a neuronal differentiation induction medium. 図６Ａ～Ｄは、本発明のヒト歯根膜由来のＳＳＥＡ－４陽性ヒト歯根膜細胞の内胚葉系組織への分化能を検討した結果を示す。図中、対照群（Ｃｏｎｔ）は分化誘導因子非添加の基本培地にて培養したものである。図６Ａは肝細胞分化誘導培地にて２１日間培養した後、アルブミンの蛍光免疫染色を行った結果を示す。6A to 6D show the results of examining the differentiation ability of human periodontal ligament cells derived from human periodontal ligament cells of the present invention into endoderm tissues. In the figure, the control group (Cont) is cultured in a basic medium to which no differentiation-inducing factor is added. FIG. 6A shows the results of fluorescent immunostaining of albumin after culturing in a hepatocyte differentiation-inducing medium for 21 days. 図６Ｂは過ヨウ素酸－シフ（ＰＡＳ）染色の結果を示す。矢頭は二核細胞の核を示す。スケールバーは５０μｍである。FIG. 6B shows the results of periodate-Schiff (PAS) staining. Arrowheads indicate binuclear cell nuclei. The scale bar is 50 μm. 図６ＣはＲＴ－ＰＣＲを用いて肝細胞マーカーの発現を調べた結果を示す。Ｈｅｐａｔｏは肝細胞分化誘導培地で培養したことを示す。FIG. 6C shows the results of examining the expression of hepatocyte markers using RT-PCR. Hepato indicates that the cells were cultured in a hepatocyte differentiation induction medium. 図６Ｄは細胞培養上清におけるアルブミン分泌のＥＬＩＳＡによる解析結果を示す。Ｈｅｐａｔｏは肝細胞分化誘導培地で培養したことを示す。FIG. 6D shows the analysis result of albumin secretion in the cell culture supernatant by ELISA. Hepato indicates that the cells were cultured in a hepatocyte differentiation induction medium.
　本発明は、第１の態様において、歯または歯周組織から細胞を得て、得られた細胞のなかからＳＳＥＡ－４陽性細胞を検出することを特徴とする、歯または歯周組織由来の幹細胞を同定する方法に関するものである。 In the first aspect, the present invention provides a stem cell derived from a tooth or periodontal tissue, wherein cells are obtained from the tooth or periodontal tissue, and SSEA-4 positive cells are detected from the obtained cells. It relates to a method for identifying.
　本発明はもう１つの態様において、歯または歯周組織から細胞を得て、得られた細胞のなかからＳＳＥＡ－４陽性細胞を単離することを特徴とする、歯または歯周組織から幹細胞を得る方法に関するものである。 In another aspect, the present invention provides a stem cell from a tooth or periodontal tissue, wherein cells are obtained from the tooth or periodontal tissue, and SSEA-4 positive cells are isolated from the obtained cells. On how to get.
　ＳＳＥＡ－４は、従来からヒトの歯の幹細胞の同定に用いられているＳＴＲＯ－１よりも抗原の発現が安定しているので、より正確にかつ効率よく、歯または歯周組織由来の幹細胞を同定あるいは単離することができる。しかしながら、上述のごとく、ＳＳＥＡ－４を歯および歯周組織由来の幹細胞のマーカーとして用いられた例はなく、歯および歯周組織由来のＳＳＥＡ－４陽性幹細胞の分化能について調べた例もない。 SSEA-4 has a more stable and stable antigen expression than STRO-1, which has been used for identification of human dental stem cells. Therefore, stem cells derived from teeth or periodontal tissues can be obtained more accurately and efficiently. Can be identified or isolated. However, as described above, there is no example in which SSEA-4 is used as a marker for stem cells derived from teeth and periodontal tissues, and there is no example in which the differentiation potential of SSEA-4 positive stem cells derived from teeth and periodontal tissues is examined.
　本発明の上記方法における細胞源は歯または歯周組織であり、例えば、歯根膜・歯髄・セメント質・歯槽骨・歯肉・歯胚などの組織が細胞源となりうる。抜歯等の歯科的処置などにより比較的得やすいことから、細胞源として歯根膜が好ましい。歯から歯根膜を得る方法は公知であり、例えば、洗浄した歯から外科用メスを用いて歯根膜を採取することができる。他の組織の取得手段、方法も公知である。 The cell source in the above method of the present invention is a tooth or periodontal tissue, and for example, tissues such as periodontal ligament, dental pulp, cementum, alveolar bone, gingiva, and tooth germ can be the cell source. Periodontal ligament is preferred as the cell source because it is relatively easy to obtain by dental treatment such as extraction. A method for obtaining a periodontal ligament from a tooth is known. For example, a periodontal ligament can be collected from a washed tooth using a scalpel. Other tissue acquisition means and methods are also known.
　得られた組織を消化して細胞を得ることができる。組織の消化方法も公知であり、例えば、蛋白分解酵素を含む溶液中で消化を行うことができる。 The cells can be obtained by digesting the obtained tissue. Tissue digestion methods are also known, and for example, digestion can be performed in a solution containing a proteolytic enzyme.
　次いで、得られた細胞のなかからＳＳＥＡ－４陽性細胞を検出および／または選別および／または同定する。これらのプロセスを行うために、フローサイトメトリーや磁気ビーズ法などの様々な方法が公知であり、本発明に適用することができる。好ましい方法はフローサイトメトリーである。細胞をフローサイトメトリーに供する前に、ＳＳＥＡ－４に特異的な物質にて細胞を標識あるいは染色しておく。細胞の標識あるいは染色に一般的に用いられる方法としては抗体染色法が例示される。好ましくは、細胞のＳＳＥＡ－４に特異的な一次抗体と、検出可能な蛍光標識を付した二次抗体の組合せを用いて細胞を染色し、フローサイトメトリーに供する。染色された細胞の解析も様々な方法、機器により行うことができるが、フローサイトメーターを用いるのが一般的である。フローサイトメーターを用いることは、本発明において目的とするＳＳＥＡ－４陽性細胞を検出および／または選別および／または同定するための好ましい手段・方法である。 Next, SSEA-4 positive cells are detected and / or sorted and / or identified from the obtained cells. In order to carry out these processes, various methods such as flow cytometry and magnetic bead method are known and can be applied to the present invention. A preferred method is flow cytometry. Prior to subjecting the cells to flow cytometry, the cells are labeled or stained with a substance specific for SSEA-4. An antibody staining method is exemplified as a method generally used for labeling or staining of cells. Preferably, the cells are stained with a combination of a primary antibody specific for the cell's SSEA-4 and a secondary antibody with a detectable fluorescent label and subjected to flow cytometry. Although the stained cells can be analyzed by various methods and instruments, a flow cytometer is generally used. The use of a flow cytometer is a preferable means and method for detecting and / or sorting and / or identifying the SSEA-4 positive cells of interest in the present invention.
　上記のようにして選別・同定されたＳＳＥＡ－４陽性細胞を、公知の方法を用いて単離することができる。細胞の単離方法としては、フローサイトメトリーや磁気ビーズ法などがあるが、フローサイトメーターを用いれば、より正確に目的のＳＳＥＡ－４陽性細胞を単離することができる。 SSEA-4 positive cells selected and identified as described above can be isolated using a known method. Examples of the cell isolation method include flow cytometry and magnetic bead method. If a flow cytometer is used, target SSEA-4-positive cells can be isolated more accurately.
　さらなる態様において、本発明は、歯または歯周組織から得られる、ＳＳＥＡ－４陽性幹細胞に関するものである。好ましくは、歯周組織は歯根膜である。好ましくは、本発明により得られるＳＳＥＡ－４陽性細胞は単離されたものである。このような細胞は、上記の本発明の方法を用いてＳＳＥＡ－４をマーカーとして得ることができる。本発明により得られる歯または歯周組織由来の幹細胞は元来増殖能が高いことに加えて、中胚葉のみならず、外胚葉・内胚葉由来のいずれの組織へも分化できる高い多分化能を有することが特色である。 In a further aspect, the present invention relates to SSEA-4 positive stem cells obtained from teeth or periodontal tissue. Preferably, the periodontal tissue is the periodontal ligament. Preferably, the SSEA-4 positive cells obtained according to the present invention are isolated. Such cells can be obtained using SSEA-4 as a marker using the method of the present invention described above. Tooth or periodontal tissue-derived stem cells obtained according to the present invention have a high pluripotency capable of differentiating not only into the mesoderm but also into any tissue derived from the ectoderm / endoderm. It is characteristic to have.
　したがって、本発明は、さらなる態様において、ＳＳＥＡ－４陽性幹細胞を分化させることを特徴とする外胚葉由来組織および／または中胚葉由来組織および／または内胚葉由来組織の製造方法を提供する。これらの方法に用いる好ましいＳＳＥＡ－４陽性幹細胞は歯または歯周組織から本発明により得られるものである。ＳＳＥＡ－４陽性幹細胞を外胚葉由来組織、中胚葉由来組織または内胚葉由来組織へと分化させるための条件は、当業者に公知である。さらに本発明は、上記製造方法により得られる組織も包含する。 Therefore, in a further aspect, the present invention provides a method for producing an ectoderm-derived tissue and / or mesoderm-derived tissue and / or endoderm-derived tissue, characterized in that SSEA-4 positive stem cells are differentiated. Preferred SSEA-4-positive stem cells used in these methods are those obtained from the teeth or periodontal tissue according to the present invention. Conditions for differentiating SSEA-4 positive stem cells into ectoderm-derived tissue, mesoderm-derived tissue or endoderm-derived tissue are known to those skilled in the art. Furthermore, this invention also includes the structure | tissue obtained by the said manufacturing method.
　本発明のＳＳＥＡ－４陽性細胞が外胚葉由来組織、中胚葉由来組織、内胚葉由来組織のいずれにも分化しうるという特色は、発生学的研究およびその他の研究分野ならびに再生医療の分野において非常に有用である。例えば、歯あるいは歯周組織が欠損した患者において、親知らずの抜歯後に歯根膜からＳＳＥＡ－４陽性幹細胞を単離し、これを歯あるいは歯周組織に分化させることにより、欠損した歯あるいは歯周組織を再生させることもできる。したがって、本発明は、歯根膜からＳＳＥＡ－４陽性幹細胞を単離し、これを歯あるいは歯周組織に分化させ、分化組織を患部に移植することを特徴とする、欠損した歯あるいは歯周組織の再生方法を提供する。また本発明は、欠損した歯あるいは歯周組織の再生に用いられる歯あるいは歯周組織を製造するための、歯根膜由来のＳＳＥＡ－４陽性細胞の使用を提供する。さらに本発明は、欠損した歯あるいは歯周組織の再生に用いられる歯あるいは歯周組織の製造方法であって、歯根膜由来のＳＳＥＡ－４陽性細胞を分化培地にて歯あるいは歯周組織へと分化させることを特徴とする方法を提供する。根膜由来のＳＳＥＡ－４陽性細胞を分化培地にて歯あるいは歯周組織へと分化させるための条件は当業者に公知であり、適宜選択して用いることができる。 The feature that the SSEA-4 positive cells of the present invention can differentiate into any of ectoderm-derived tissue, mesoderm-derived tissue, and endoderm-derived tissue is very important in developmental research and other research fields as well as in the field of regenerative medicine. Useful for. For example, in a patient with missing teeth or periodontal tissue, SSEA-4 positive stem cells are isolated from the periodontal ligament after wisdom tooth extraction, and differentiated into teeth or periodontal tissue, so that the missing tooth or periodontal tissue It can also be played back. Therefore, the present invention isolates SSEA-4 positive stem cells from the periodontal ligament, differentiates them into teeth or periodontal tissue, and transplants the differentiated tissue to the affected area. Provide a playback method. The present invention also provides the use of periodontal ligament-derived SSEA-4 positive cells for producing a tooth or periodontal tissue used for regeneration of a missing tooth or periodontal tissue. Furthermore, the present invention relates to a method for producing a tooth or periodontal tissue used for regeneration of a missing tooth or periodontal tissue, wherein periodontal ligament-derived SSEA-4 positive cells are transformed into the tooth or periodontal tissue in a differentiation medium. A method characterized by differentiating is provided. Conditions for differentiating SSEA-4 positive cells derived from the basement membrane into teeth or periodontal tissues in a differentiation medium are known to those skilled in the art and can be appropriately selected and used.
　以下に実施例を示して本発明をさらに詳細かつ具体的に説明するが、実施例は本発明を限定するものと解釈してはならない。 Hereinafter, the present invention will be described in more detail and specifically with reference to examples. However, the examples should not be construed as limiting the present invention.
　実施例１：歯根膜からの幹細胞の同定および単離、ならびに得られた幹細胞における各種因子の発現 Example 1: Identification and isolation of stem cells from periodontal ligament and expression of various factors in the obtained stem cells
　本研究は、岡山大学大学院医歯薬学総合研究科倫理委員会の承認（承認番号３６５）のもとに行われた。本研究に用いたヒト歯根膜は、矯正治療を目的に便宜抜去された患者の健全歯より採取した。歯根膜の提供を受けるに際して、規定に基づきこの歯根膜の使用目的を十分に説明し、同意を得たうえで本研究を開始した。 This research was conducted with the approval of the Okayama University Graduate School of Biomedical Sciences Ethics Committee (approval number 365). The human periodontal ligament used in this study was collected from the healthy teeth of patients who were removed for the purpose of orthodontic treatment. Upon receiving the periodontal ligament, we fully explained the purpose of use of the periodontal ligament based on the regulations, and obtained this consent before starting this study.
　提供を受けた患者の歯をＣａ２＋、Ｍｇ２＋非含有リン酸緩衝液（phosphate-buffered saline: PBS(-)）で２回洗浄した後、外科用替え刃メスを使用して歯根表面を削がないよう歯根膜を採取した。得られた歯根膜は、５ｍｇ／ｍＬ　コラゲナーゼ　タイプＩＩ（Worthington, Lakewood, NJ, USA）および２．５ｍｇ／ｍＬ　ディスパーゼ　Ｉ（Roche, Penzbeg, Germany）含有低グルコースダルベッコ変法イーグル培地（DMEM; Invitrogen, Carlsbad, CA, USA）にて３７℃で６０分間消化した。得られたヒト歯根膜細胞は、１５％非働化済ウシ胎仔血清（heat inactivated fetal bovine serum: HIFBS; HyClone, Logan, UT, USA）および１％　ペニシリン／ストレプトマイシン（最終濃度各々１００Ｕ／ｍＬおよび１００μｇ／ｍＬ；Invitrogen）含有低グルコースＤＭＥＭで培養した。培地は週２回、半量を交換した。
　上記条件下、プラスチック培養皿にて培養したヒト歯根膜細胞を位相差顕微鏡にて観察した。細胞は紡錘形で単核の繊維芽細胞様の形態を呈しており、形態的には骨髄間葉系幹細胞と同じであった。培養２４時間後には、細胞の対称分裂が認められた（図１左上および左下パネル）。培養７日後には、網目状ネットワークを形成した（図１右上および右下パネル）。 The patient's teeth were washed twice with Ca 2+ , Mg 2+ -free phosphate buffer (phosphate-buffered saline: PBS (-)), and then the root surface was shaved using a surgical blade. Periodontal ligaments were collected so that there was no damage. The periodontal ligaments obtained were modified with low glucose Dulbecco's modified Eagle medium (DMEM; Invitrogen, containing 5 mg / mL collagenase type II (Worthington, Lakewood, NJ, USA) and 2.5 mg / mL dispase I (Roche, Penzbeg, Germany). Carlsbad, CA, USA) at 37 ° C. for 60 minutes. The resulting human periodontal ligament cells were treated with 15% heat inactivated fetal bovine serum (HIFBS; HyClone, Logan, UT, USA) and 1% penicillin / streptomycin (final concentrations of 100 U / mL and 100 μg / 100%, respectively). Incubated with low glucose DMEM containing mL; Invitrogen). Half of the medium was changed twice a week.
Under the above conditions, human periodontal ligament cells cultured in a plastic culture dish were observed with a phase contrast microscope. The cells were spindle-shaped and had a mononuclear fibroblast-like morphology and were morphologically the same as bone marrow mesenchymal stem cells. After 24 hours of culture, symmetric cell division was observed (FIG. 1, upper left and lower left panels). After 7 days of culture, a mesh network was formed (upper right and lower right panels in FIG. 1).
　フローサイトメトリーを用いて、培養ヒト歯根膜細胞における各種抗原の発現を解析した。フローサイトメトリーの手順は以下のとおりであった。 The expression of various antigens in cultured human periodontal ligament cells was analyzed using flow cytometry. The flow cytometry procedure was as follows.
　細胞は２％　ＨＩＦＢＳ、１０ｍＭ　ＨＥＰＥＳ（Invitrogen）および１％　ペニシリン／ストレプトマイシン含有ハンクス平衡緩衝塩溶液（HBSS; Invitrogen）に再懸濁した。細胞内抗原の検出の際は、細胞を４％　パラホルムアルデヒド（PFA; Electron Microscopy Sciences, Hatfield, PA, USA）にて５分間固定を行った後、０．１％　ノニデット　Ｐ－４０（Nacalai Tesque, Kyoto, Japan）にて５分間透過化処理を行った。 Cells were resuspended in 2% HIFBS, 10 mM HEPES (Invitrogen) and 1% penicillin / streptomycin-containing Hank's balanced buffer solution (HBSS; Invitrogen). When detecting intracellular antigens, cells were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA, USA) for 5 minutes, and then 0.1% Nonidet P-40 (Nacalai Tesque, Permeabilization treatment was performed for 5 minutes at Kyoto, Sakai Japan).
　抗体染色の場合、細胞懸濁液に一次抗体を添加し、６０分間氷上にて反応させた。未標識一次抗体を用いる場合は、一次抗体反応後さらに蛍光標識された二次抗体を添加し、３０分間氷上にて反応させた。死細胞の検出には、７－アミノ－アクチノマイシンＤ（7-AAD;BD Biosciences, San Jose, CA, USA）を用いた。 In the case of antibody staining, the primary antibody was added to the cell suspension and allowed to react on ice for 60 minutes. In the case of using an unlabeled primary antibody, a fluorescently labeled secondary antibody was further added after the primary antibody reaction and allowed to react on ice for 30 minutes. For detection of dead cells, 7-amino-actinomycin D (7-AAD; BD Biosciences, San San Jose, CA, USA) was used.
　染色した細胞は、ＦＡＣＳＡｒｉａ（BD Biosciences）を用いて解析した。フルオレセインイソチオシアネート（FITC）、フィコエリスリン（PE）、７－ＡＡＤおよびＰＥ－Ｃｙ７は、４８８ｎｍのアルゴンイオンレーザーで励起し、それぞれ５３０／３０、５７５／２６、６９５／４０、７８０／６０のフィルターを用いて検出した。アロフィコシアニン（APC）は６３３ｎｍのＨｅ－Ｎｅレーザーで励起し、６６０／２０のフィルターで検出した。死細胞は前方・側方散乱光上でのゲーティング、および７－ＡＡＤ陽性集団を除くことによって除外した。データ解析にはＦｌｏｗＪｏ（Tree Star, Ashland, OR, USA）を用いた。 Stained cells were analyzed using FACSAria (BD Biosciences). Fluorescein isothiocyanate (FITC), phycoerythrin (PE), 7-AAD, and PE-Cy7 are excited by a 488 nm argon ion laser and have 530/30, 575/26, 695/40, and 780/60 filters, respectively. It detected using. Allophycocyanin (APC) was excited with a 633 nm He-Ne laser and detected with a 660/20 filter. Dead cells were excluded by gating on forward and side scatter and removing the 7-AAD positive population. FlowJo (Tree Star, Ashland, OR, USA) was used for data analysis.
　フローサイトメトリーの結果を図２に示す。培養ヒト歯根膜細胞は、ＣＤ２９、ＣＤ４４、ＣＤ７１、ＣＤ７３、ＣＤ９０、ＣＤ１０５を発現するなど、骨髄間葉系幹細胞と同様の抗原の発現パターンを呈していた。 Fig. 2 shows the results of flow cytometry. Cultured human periodontal ligament cells exhibited the same antigen expression pattern as bone marrow mesenchymal stem cells, such as expressing CD29, CD44, CD71, CD73, CD90, and CD105.
　次に、培養ヒト歯根膜細胞における胚性幹細胞関連分子の発現について調べた。用いた手法は、蛍光免疫細胞化学、アルカリフォスファターゼ活性の検出、フローサイトメトリー、テロメア長の測定、および細胞増殖能の評価であった。これらの実験手順を以下に説明する（フローサイトメトリーは上で説明した）。 Next, the expression of embryonic stem cell-related molecules in cultured human periodontal ligament cells was examined. The procedures used were fluorescence immunocytochemistry, detection of alkaline phosphatase activity, flow cytometry, telomere length measurement, and evaluation of cell proliferation ability. These experimental procedures are described below (flow cytometry was described above).
　蛍光免疫細胞化学
　細胞は４％　ＰＦＡにより１０分間固定した後、０．１％　ノニデットＰ－４０および１０％正常ヤギ血清（Sigma）含有ＰＢＳ（－）を用いて３０分間透過化処理およびブロッキングを行った。一次抗体は室温にて６０分間、二次抗体は室温にて３０分間、それぞれ反応させた。核染色は、４’，６－ジアミジノ－２－フェニルインドール（DAPI; Sigma）を用いて行った。染色した細胞は蛍光顕微鏡下にて観察を行った。 Fluorescent immunocytochemistry Cells were fixed with 4% PFA for 10 minutes, then permeabilized and blocked for 30 minutes with PBS (-) containing 0.1% nonidet P-40 and 10% normal goat serum (Sigma) It was. The primary antibody was reacted at room temperature for 60 minutes, and the secondary antibody was reacted at room temperature for 30 minutes. Nuclear staining was performed using 4 ′, 6-diamidino-2-phenylindole (DAPI; Sigma). The stained cells were observed under a fluorescence microscope.
　アルカリフォスファターゼ活性の検出
　アルカリフォスファターゼ活性の検出は、Alkaline Phosphatase Detection Kit（Chemicon, Temecula, CA, USA）を用いて行った。 Detection of alkaline phosphatase activity Alkaline phosphatase activity was detected using the Alkaline Phosphatase Detection Kit (Chemicon, Temecula, CA, USA).
　テロメア長の測定
　テロメア長の測定は、Telomere PNA Kit/FITC for Flow Cytometry（Dako, Glostrup, Denmark）を用いて行った。 Measurement of telomere length The telomere length was measured using the Telomere PNA Kit / FITC for Flow Cytometry (Dako, Glostrup, Denmark).
　細胞増殖能の評価
　細胞を、フローサイトメトリーを用いて３００個／ウェルの濃度で９６－ウェル培養プレートに播種し、培養を行った。培養７日目に、Cell Titer 96 AQueous（Promega, Madison, WI, USA）を用いて細胞増殖を測定した。 Evaluation of Cell Proliferation Capacity Cells were seeded in a 96-well culture plate at a concentration of 300 cells / well using flow cytometry and cultured. On day 7 of culture, cell proliferation was measured using Cell Titer 96 AQ ueous (Promega, Madison, Wis., USA).
　結果を図３に示す。培養ヒト歯根膜細胞は、ヒト胚性幹細胞（ＥＳ細胞）のマーカー分子を発現していた。これらの結果より、ＳＳＥＡ－４を用いて、未分化なヒト歯根膜幹細胞を同定できることがわかった。 The results are shown in FIG. The cultured human periodontal ligament cells expressed a marker molecule of human embryonic stem cells (ES cells). From these results, it was found that undifferentiated human periodontal ligament stem cells can be identified using SSEA-4.
　免疫蛍光染色の結果を図３Ａに示す。本発明により得られた幹細胞においてＥＳ細胞関連分子（表面抗原分子：ＳＳＥＡ－１、ＳＳＥＡ－３、ＳＳＥＡ－４、ＴＲＡ－１－６０、ＴＲＡ－１－８１、および転写因子：ＯＣＴ４、ＮＡＮＯＧ、ＳＯＸ２、ＲＥＸ１）の発現が確認された。また、本発明により得られた幹細胞においてアルカリフォスファターゼ（ＡＬＰ）活性も検出された。 The result of immunofluorescence staining is shown in FIG. 3A. In stem cells obtained by the present invention, ES cell-related molecules (surface antigen molecules: SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and transcription factors: OCT4, NANOG, SOX2 , REX1) expression was confirmed. Moreover, alkaline phosphatase (ALP) activity was also detected in the stem cells obtained by the present invention.
　フローサイトメトリーを用いて培養ヒト歯根膜細胞におけるＳＳＥＡ－１、ＳＳＥＡ－３およびＳＳＥＡ－４の発現を調べた結果を図３Ｂに示す。ＳＳＥＡ－４の発現が多くみとめられた。 FIG. 3B shows the results of examining the expression of SSEA-1, SSEA-3, and SSEA-4 in cultured human periodontal ligament cells using flow cytometry. Many expression of SSEA-4 was observed.
　さらに、レチノイン酸を用いて、培養ヒト歯根膜細胞を分化誘導した時の、ＳＳＥＡ－１、ＳＳＥＡ－３、ＳＳＥＡ－４抗原発現の変化を調べた。培地に１０－５Ｍ　オールトランスレチノイン酸を添加し、７日間培養した。結果を図３Ｃに示す。未分化な状態では、グロボ系糖脂質（ＳＳＥＡ－３・ＳＳＥＡ－４）の発現が高く、ラクト系糖脂質（ＳＳＥＡ－１）の発現が低かった。レチノイン酸で分化誘導を引き起こすと、グロボ系糖脂質の発現が下がり、ラクト系糖脂質の発現が上昇した。このように、ヒトＥＳ細胞と同様、ヒト歯根膜細胞もその分化状態に応じて細胞膜糖脂質の変化が起こることがわかった。 Furthermore, changes in SSEA-1, SSEA-3, and SSEA-4 antigen expression when cultured human periodontal ligament cells were induced to differentiate using retinoic acid were examined. 10 −5 M all-trans retinoic acid was added to the medium and cultured for 7 days. The results are shown in FIG. 3C. In an undifferentiated state, the expression of globo glycolipid (SSEA-3 / SSEA-4) was high and the expression of lacto glycolipid (SSEA-1) was low. When differentiation induction was caused by retinoic acid, the expression of globo glycolipid decreased and the expression of lacto glycolipid increased. Thus, like human ES cells, human periodontal ligament cells were found to undergo changes in cell membrane glycolipids depending on their differentiation state.
　培養ヒト歯根膜細胞における、ＳＳＥＡ－４陽性細胞と陰性細胞のテロメア長の比較を行った結果を図３Ｄに示す。ＳＳＥＡ－４陽性細胞はテロメア長が長く、ＳＳＥＡ－４陰性細胞は短いことがわかった。 FIG. 3D shows the results of comparison of the telomere length of SSEA-4 positive cells and negative cells in cultured human periodontal ligament cells. It was found that SSEA-4 positive cells had a long telomere length and SSEA-4 negative cells were short.
　培養ヒト歯根膜細胞における、ＳＳＥＡ－４陽性細胞と陰性細胞の増殖能の比較を行った結果を図３Ｅに示す。ＳＳＥＡ－４陽性細胞は増殖能が高く、ＳＳＥＡ－４陰性細胞は低かった。 FIG. 3E shows the results of comparison of the proliferation ability of SSEA-4 positive cells and negative cells in cultured human periodontal ligament cells. SSEA-4 positive cells were highly proliferative and SSEA-4 negative cells were low.
実施例２：ヒト歯根膜由来のＳＳＥＡ－４陽性幹細胞の分化能
　本発明のヒト歯根膜由来のＳＳＥＡ－４陽性幹細胞の中胚葉系組織、外胚葉系組織および内胚葉系組織への分化能について、インビトロにて調べた。実験手順を以下に示す。 Example 2: Differentiation ability of human periodontal ligament-derived SSEA-4-positive stem cells Differentiation ability of human periodontal ligament-derived SSEA-4 positive stem cells of the present invention into mesodermal, ectoderm, and endoderm tissues Investigated in vitro. The experimental procedure is shown below.
　培養ヒト歯根膜細胞より、フローサイトメトリーを用いてＳＳＥＡ－４陽性細胞の単一クローン細胞を単離し、以下の分化誘導を行った。 Single clone cells of SSEA-4 positive cells were isolated from cultured human periodontal ligament cells using flow cytometry, and the following differentiation induction was performed.
　脂肪細胞への分化誘導は、１０％　ＨＩＦＢＳ、１％　ペニシリン／ストレプトマイシン、１μＭ　デキサメタソン（Sigma）、０．２ｍＭ　インドメタシン（Sigma）、０．０１ｍｇ／ｍＬ　インスリン（Sigma）、０．５ｍＭ　３－イソブチル－１－メチルキサンチン（Sigma）含有高グルコースＤＭＥＭ（Invitrogen）にて２１日間培養を行った。培養した細胞は、オイルレッドＯ（Sigma）にて染色し光学顕微鏡下において観察を行った。細胞培養上清におけるアジポネクチンの分泌は、Human Adiponectin/Acrp30 Immunoassay（R&D Systems, Minneapolis, MN, USA）を用いて測定した。 Induction of differentiation into adipocytes is 10% HIFBS, 1% penicillin / streptomycin, 1 μM dexamethasone (Sigma), 0.2 mM indomethacin (Sigma), 0.01 mg / mL insulin (Sigma), 0.5 mM 3-isobutyl-1 -Cultured in high glucose DMEM (Invitrogen) containing methylxanthine (Sigma) for 21 days. The cultured cells were stained with oil red O (Sigma) and observed under an optical microscope. Secretion of adiponectin in the cell culture supernatant was measured using Human® Adiponectin / Acrp30® Immunoassay (R & D® Systems, Minneapolis, MN, USA).
　骨芽細胞への分化誘導は、１０％　ＨＩＦＢＳ、１％　ペニシリン／ストレプトマイシン、１００ｎＭ　デキサメタソン、０．０５ｍＭ　Ｌ－アスコルビン酸（Sigma）、１０ｍＭ　β－グリセロリン酸（Sigma）含有最小必須アルファ培地（α-MEM; Invitrogen）にて２１日間培養を行った。培養した細胞は、アリザリンレッドＳ（Sigma）にて染色し光学顕微鏡下において観察を行った。細胞に沈着したカルシウムの測定は、Calcium E-Test Wako（Wako Pure Chemical, Tokyo, Japan）を用いて測定した。 Induction of differentiation into osteoblasts is minimal essential alpha medium (α-MEM containing 10% HIFBS, 1% penicillin / streptomycin, 100 nM dexamethasone, 0.05 mM L-ascorbic acid (Sigma), 10 mM β-glycerophosphate (Sigma). Culturing was carried out for 21 days in Invitrogen). The cultured cells were stained with alizarin red S (Sigma) and observed under an optical microscope. Calcium deposited on the cells was measured using Calcium E-Test Wako (Wako Pure Chemical, Tokyo, Japan).
　軟骨細胞への分化誘導は、まず３Ｘ１０５の細胞を１５ｍＬ　ポリプロピレン製コニカルチューブ（Corning, Corning, NY, USA）に分注し、遠心して細胞塊を形成した。この細胞塊を１％　ペニシリン／ストレプトマイシン、１００ｎＭ　デキサメタソン、１ｍＭ　ピルビン酸ナトリウム（Sigma）、０．１７ｍＭ　Ｌ－アスコルビン酸、０．３５ｍＭ　Ｌ－プロリン（Sigma）、ITS + Universal Culture Supplement Premix(BD Biosciences）、１０ｎｇ／ｍＬ　ＴＧＦ－β３（R&D Systems）含有高グルコースＤＭＥＭにて２１日間培養を行った。培養した細胞は、パラフィン包埋して切片を作成した後、アルシアンブルー（Sigma）にて染色し光学顕微鏡下において観察を行った。 For induction of differentiation into chondrocytes, 3 × 10 5 cells were first dispensed into a 15 mL polypropylene conical tube (Corning, Corning, NY, USA) and centrifuged to form a cell mass. This cell mass was divided into 1% penicillin / streptomycin, 100 nM dexamethasone, 1 mM sodium pyruvate (Sigma), 0.17 mM L-ascorbic acid, 0.35 mM L-proline (Sigma), ITS + Universal Culture Supplement Premix (BD Biosciences), The cells were cultured for 21 days in high glucose DMEM containing 10 ng / mL TGF-β3 (R & D Systems). The cultured cells were embedded in paraffin to prepare sections, stained with Alcian blue (Sigma), and observed under an optical microscope.
　神経細胞への分化誘導は、１５％　ＨＩＦＢＳ、１％　ペニシリン／ストレプトマイシン、１０ｎｇ／ｍＬ　ｂＦＧＦ（R&D Systems, Minneapolis, MN, USA）含有低グルコースＤＭＥＭにて２４時間培養した後、２％　ジメチルスルホキシド（DMSO; Sigma）、１００μＭ　ブチレート化ヒドロキシアニソール（BHA; Sigma）、１０μＭ　フォルスコリン（Sigma）、５Ｕ／ｍＬ　ヘパリン（Sigma）、５ｎＭ　Ｋ２５２ａ（Sigma）、２５ｍＭ　ＫＣｌ（Sigma）、２ｍＭ　２－プロピルペンタン酸（Sigma）、１Ｘ　Ｎ２サプリメント（Invitrogen）、１０ｎｇ／ｍＬ　ヒト塩基性線維芽細胞増殖因子（bFGF; R&D Systems）、１０ｎｇ／ｍＬ　ヒト血小板由来増殖因子ＢＢ（PDGF-BB; R&D Systems）含有低グルコースＤＭＥＭにて４８時間培養した。培養した細胞は、抗ニューロン－特異的エノラーゼ（ＮＳＥ）抗体およびAlexa Fluor 594標識二次抗体を用いて染色し蛍光顕微鏡下にて観察を行った。 Induction of differentiation into nerve cells was performed by culturing for 24 hours in low glucose DMEM containing 15% HIFBS, 1% penicillin / streptomycin, 10 ng / mL bFGF (R & D Systems, polisMinneapolis, MN, USA), and then 2% dimethyl sulfoxide (DMSO). ; Sigma), 100 μM butyrated hydroxyanisole (BHA; Sigma), 10 μM forskolin (Sigma), 5 U / mL heparin (Sigma), 5 nM K252a (Sigma), 25 mM KCl (Sigma), 2 mM 2-propylpentanoic acid (Sigma) ) Low glucose DMEM containing 1X N2 supplement (Invitrogen), 10 ng / mL human basic fibroblast growth factor (bFGF; R & D Systems), 10 ng / mL human platelet-derived growth factor BB (PDGF-BB; R & D Systems) Cultured for 48 hours. The cultured cells were stained with an anti-neuron-specific enolase (NSE) antibody and an Alexa Fluor 594 labeled secondary antibody and observed under a fluorescence microscope.
　肝細胞への分化誘導は、第１段階として２％　ＨＩＦＢＳ、１％　ペニシリン／ストレプトマイシン、１００ｎｇ／ｍＬ　ａＦＧＦ（R&D Systems）含有低グルコースＤＭＥＭにて５日間培養、第２段階として２％　ＨＩＦＢＳ、１％　ペニシリン／ストレプトマイシン、２０ｎｇ／ｍＬ　ＨＧＦ（R&D Systems）含有低グルコースＤＭＥＭにて５日間培養した後、第３段階として２％　ＨＩＦＢＳ、１％　ペニシリン／ストレプトマイシン、２０ｎｇ／ｍＬ　ＨＧＦ、１０ｎＭ　デキサメタソン、1X insulin-transferrin-selenium-X（Invitrogen）、１０ｎｇ／ｍＬ　オンコスタチンＭ（R&D Systems）含有低グルコースＤＭＥＭにて１１日間培養した。培養した細胞は、抗アルブミン抗体およびAlexa Fluor 594標識二次抗体を用いて染色し蛍光顕微鏡下にて観察を行った。細胞内グリコーゲンは、Periodic acid-schiff (PAS) staining kit（Sigma）を用いて検出した。細胞培養上清におけるアルブミンの分泌は、Human serum albumin assay（Cygnous Technologies, Southport, NC, USA）を用いて測定した。 For induction of differentiation into hepatocytes, the first stage is 2% HIFBS, 1% penicillin / streptomycin, 100 ng / mL aFGF (R & D Systems) -containing low glucose DMEM for 5 days, the second stage is 2% HIFBS, 1% After culturing for 5 days in penicillin / streptomycin, 20 ng / mL HGF (R & D® Systems) -containing low glucose DMEM, the third stage is 2% HIFBS, 1% penicillin / streptomycin, 20 ng / mL HGF, 10 nM dexamethasone, 1X insulin-transferrin -selenium-X (Invitrogen), 10 ng / mL Oncostatin M (R & D Systems) -containing low glucose DMEM was cultured for 11 days. The cultured cells were stained with an anti-albumin antibody and an Alexa-Fluor-594 labeled secondary antibody and observed under a fluorescence microscope. Intracellular glycogen was detected using Periodic acid-schiff (PAS) holding kit (Sigma). Albumin secretion in the cell culture supernatant was measured using Human® serum® albumin assay (Cygnous Technologies, Southport, NC, USA).
　本発明のヒト歯根膜由来のＳＳＥＡ－４陽性幹細胞の中胚葉系組織への分化能を検討した結果を図４Ａ～Ｉに、外胚葉系組織への分化能を検討した結果を図５Ａ～Ｂに、そして内胚葉系組織への分化能を検討した結果を図６Ａ～Ｄに示す。６３．６％（１４／２２）のＳＳＥＡ－４陽性幹細胞株が多分化能を有していることが示された。 4A to I show the results of examining the differentiation ability of human periodontal ligament-derived SSEA-4 positive stem cells of the present invention into mesodermal tissues, and FIGS. 5A to B show the results of examination of differentiation ability into ectoderm tissues. 6A to 6D show the results of examining the differentiation ability into endoderm tissues. 63.6% (14/22) of SSEA-4 positive stem cell lines were shown to have pluripotency.
　ＳＳＥＡ－４陽性幹細胞を脂肪細胞分化誘導培地にて２１日間培養した後、以下の解析を行った。図４Ａに示すように、オイルレッドＯ染色を行ったところ、染色像が確認された。図４Ｂに示すように、ＲＴ－ＰＣＲにより脂肪細胞マーカーの発現を調べたところ、ＰＰＡＲ－γ、ＡＬＢＰ、ＬＰＬといった脂肪細胞マーカーの発現が確認された。図４Ｃに示すように、オイルレッドＯ染色による脂肪滴形成の定量的解析を行ったところ、対照群と比較して約５倍のオイルレッドＯ染色性がみとめられた。図４Ｄに示すように、ＥＬＩＳＡにより細胞培養上清におけるアジポネクチン分泌の解析を行ったところ、その分泌が経時的に増加することが確認された。 SSEA-4 positive stem cells were cultured in an adipocyte differentiation induction medium for 21 days, and then analyzed as follows. As shown in FIG. 4A, when oil red O staining was performed, a stained image was confirmed. As shown in FIG. 4B, the expression of adipocyte markers such as PPAR-γ, ALBP, and LPL was confirmed by examining the expression of adipocyte markers by RT-PCR. As shown in FIG. 4C, when quantitative analysis of lipid droplet formation by oil red O staining was performed, about 5 times as much oil red O staining as the control group was observed. As shown in FIG. 4D, when adiponectin secretion was analyzed in the cell culture supernatant by ELISA, it was confirmed that the secretion increased with time.
　ＳＳＥＡ－４陽性幹細胞を骨芽細胞分化誘導培地にて２１日間培養した後、以下の解析を行った。図４Ｅに示すように、アリザリンレッドＳ染色を行ったところ、染色像が確認された。図４Ｆに示すように、ＲＴ－ＰＣＲにより骨芽細胞マーカーの発現を調べたところ、ＯＰＮ、ＢＳＰといった骨芽細胞マーカーの発現が確認された。図４Ｇに示すように、細胞に沈着したＣａの定量的解析を行ったところ、沈着Ｃａ量が経時的に増加することが確認された。 SSEA-4 positive stem cells were cultured in an osteoblast differentiation induction medium for 21 days, and then analyzed as follows. As shown in FIG. 4E, when alizarin red S staining was performed, a stained image was confirmed. As shown in FIG. 4F, when the expression of osteoblast markers was examined by RT-PCR, the expression of osteoblast markers such as OPN and BSP was confirmed. As shown in FIG. 4G, when quantitative analysis of Ca deposited on the cells was performed, it was confirmed that the amount of deposited Ca increased with time.
　ＳＳＥＡ－４陽性幹細胞を軟骨細胞分化誘導培地にて２１日間培養した後、以下の解析を行った。図４Ｈに示すように、パラフィン包埋して切片作成後、アルシアンブルー染色を行ったところ、染色像が確認された。図４Ｉに示すように、ＲＴ－ＰＣＲにより軟骨細胞マーカーの発現を調べたところ、ＣＯＬ　ＩＩ、ＣＯＬ　Ｘといった軟骨細胞マーカーの発現が確認された。 SSEA-4 positive stem cells were cultured in a chondrocyte differentiation induction medium for 21 days, and then analyzed as follows. As shown in FIG. 4H, after the section was prepared by embedding in paraffin, stained with Alcian blue, a stained image was confirmed. As shown in FIG. 4I, when the expression of chondrocyte markers was examined by RT-PCR, the expression of chondrocyte markers such as COL II and COL X was confirmed.
　ＳＳＥＡ－４陽性幹細胞を神経細胞分化誘導培地にて３日間培養した後、以下の解析を行った。図５Ａに示すように、ニューロン特異的エノラーゼ（ＮＳＥ）の蛍光免疫染色を行ったところ、染色像が確認された。図５Ｂに示すように、ＲＴ－ＰＣＲにより神経細胞マーカーの発現を調べたところ、ＮＦ－Ｍ、Ｔｕｂ－β３といった神経細胞マーカーの発現が確認された。 SSEA-4 positive stem cells were cultured in a neuronal differentiation induction medium for 3 days, and then analyzed as follows. As shown in FIG. 5A, when immunofluorescent staining of neuron-specific enolase (NSE) was performed, a stained image was confirmed. As shown in FIG. 5B, the expression of neuronal markers such as NF-M and Tub-β3 was confirmed by examining the expression of neuronal markers by RT-PCR.
　ＳＳＥＡ－４陽性幹細胞を肝細胞分化誘導培地にて２１日間培養した後、以下の解析を行った。図６Ａに示すように、アルブミンの蛍光免疫染色を行ったところ、染色像が確認された。図６Ｂに示すように、過ヨウ素酸－シフ（ＰＡＳ）染色を行ったところ、染色像が確認された。図６Ｃに示すように、ＲＴ－ＰＣＲにより肝細胞マーカーの発現を調べたところ、ＡＬＢ、ＡＦＰといった幹細胞マーカーの発現が確認された。図６Ｄに示すように、ＥＬＩＳＡにより細胞培養上清におけるアルブミン分泌の解析を行ったところ、その分泌が経時的に増加することが確認された。 SSEA-4 positive stem cells were cultured in a hepatocyte differentiation-inducing medium for 21 days, and then analyzed as follows. As shown in FIG. 6A, when fluorescent immunostaining of albumin was performed, a stained image was confirmed. As shown in FIG. 6B, stained with periodic acid-Schiff (PAS), a stained image was confirmed. As shown in FIG. 6C, when the expression of hepatocyte markers was examined by RT-PCR, the expression of stem cell markers such as ALB and AFP was confirmed. As shown in FIG. 6D, when the albumin secretion in the cell culture supernatant was analyzed by ELISA, it was confirmed that the secretion increased with time.
　このように、本発明のＳＳＥＡ－４陽性幹細胞は、中胚葉系組織のみならず、外胚葉系組織、内胚葉系組織のいずれにも分化できる高い分化能を有することがわかった。 Thus, it was found that the SSEA-4 positive stem cells of the present invention have a high differentiation ability capable of differentiating not only into mesodermal tissues but also into ectodermal tissues and endoderm tissues.
　本発明によれば、歯または歯周組織由来の細胞から確実、簡便かつ効率的に幹細胞を同定し、単離することができ、得られる幹細胞は外胚葉、中胚葉および内胚葉由来のいずれの組織へも分化できる高い分化能を持つので、発生学的研究およびその他の研究分野ならびに再生医療の分野において有用である。 According to the present invention, stem cells can be reliably and easily identified and isolated from teeth or periodontal tissue-derived cells, and the obtained stem cells can be any of ectoderm, mesoderm and endoderm. Since it has a high differentiation ability capable of differentiating into tissues, it is useful in developmental research and other research fields as well as in the field of regenerative medicine.

Claims (15)

  1. 　歯または歯周組織から細胞を得て、
    　得られた細胞のなかからＳＳＥＡ－４陽性細胞を検出する
    ことを特徴とする、歯または歯周組織由来の幹細胞を同定する方法。     Get cells from teeth or periodontal tissue,
    A method for identifying stem cells derived from teeth or periodontal tissue, wherein SSEA-4 positive cells are detected from the obtained cells.
  2. 　検出がフローサイトメーターを用いて行われる請求項１記載の方法。 The method according to claim 1, wherein the detection is performed using a flow cytometer.
  3. 　歯または歯周組織から細胞を得て、
    　得られた細胞のなかからＳＳＥＡ－４陽性細胞を単離する
    ことを特徴とする、歯または歯周組織から幹細胞を得る方法。     Get cells from teeth or periodontal tissue,
    A method for obtaining stem cells from teeth or periodontal tissue, comprising isolating SSEA-4 positive cells from the obtained cells.
  4. 　単離がフローサイトメーターまたは磁気ビーズを用いて行われる請求項３記載の方法。 The method according to claim 3, wherein the isolation is performed using a flow cytometer or magnetic beads.
  5. 　歯または歯周組織から得られる、単離されたＳＳＥＡ－４陽性幹細胞。 Isolated SSEA-4 positive stem cells obtained from teeth or periodontal tissue.
  6. 　請求項３または４記載の方法により得られる、単離されたＳＳＥＡ－４陽性幹細胞。 An isolated SSEA-4 positive stem cell obtained by the method according to claim 3 or 4.
  7. 　歯根膜由来である請求項５または６記載の幹細胞。 The stem cell according to claim 5 or 6, which is derived from periodontal ligament.
  8. 　外胚葉、中胚葉および内胚葉由来のいずれの組織へも分化できる請求項５～７のいずれか１項記載の幹細胞。 The stem cell according to any one of claims 5 to 7, which can differentiate into any tissue derived from ectoderm, mesoderm and endoderm.
  9. 　請求項５～７のいずれか１項記載の幹細胞を分化させることを特徴とする、外胚葉由来組織の製造方法。 A method for producing an ectoderm-derived tissue, wherein the stem cell according to any one of claims 5 to 7 is differentiated.
  10. 　請求項５～７のいずれか１項記載の幹細胞を分化させることを特徴とする、中胚葉由来組織の製造方法。 A method for producing a mesoderm-derived tissue, wherein the stem cell according to any one of claims 5 to 7 is differentiated.
  11. 　請求項５～７のいずれか１項記載の幹細胞を分化させることを特徴とする、内胚葉由来組織の製造方法。 A method for producing an endoderm-derived tissue, wherein the stem cell according to any one of claims 5 to 7 is differentiated.
  12. 　請求項９～１１のいずれか１項記載の方法により得られる組織。 A tissue obtained by the method according to any one of claims 9 to 11.
  13. 　歯根膜からＳＳＥＡ－４陽性幹細胞を単離し、これを歯あるいは歯周組織に分化させ、分化組織を患部に移植することを特徴とする、欠損した歯あるいは歯周組織の再生方法。 A method for regenerating a missing tooth or periodontal tissue, comprising isolating SSEA-4 positive stem cells from the periodontal ligament, differentiating the stem cells into teeth or periodontal tissue, and transplanting the differentiated tissue to the affected area.
  14. 　欠損した歯あるいは歯周組織の再生に用いられる歯あるいは歯周組織を製造するための、歯根膜由来のＳＳＥＡ－４陽性細胞の使用。 Use of periodontal ligament-derived SSEA-4 positive cells for the production of teeth or periodontal tissues used for regeneration of missing teeth or periodontal tissues.
  15. 　欠損した歯あるいは歯周組織の再生に用いられる歯あるいは歯周組織の製造方法であって、歯根膜由来のＳＳＥＡ－４陽性細胞を分化培地にて歯あるいは歯周組織へと分化させることを特徴とする方法。 A method for producing a tooth or periodontal tissue used for regeneration of a missing tooth or periodontal tissue, characterized by differentiating periodontal ligament-derived SSEA-4 positive cells into teeth or periodontal tissue in a differentiation medium And how to.
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