JP2010252778A - Method for separating dental pulp-derived mesenchymal stem cell, separated stem cell and differentiation to hepatocyte lineage using them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a separation method for easily acquiring mesenchymal stem cells from dental pulp and a method for differentiating separated cells into a hepatocyte lineage. <P>SOLUTION: Dental pulp-derived mesenchymal stem cells are separated from dental pulp of baby tooth or the last molar tooth by using an CD117 (c-kit) or CD44 antibody. The dental pulp-derived mesenchymal stem cells are cultured in a medium containing HGF, dexamethasone, oncostatin and ITS-X and differentiated to hepatocyte lineage cells. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for separating stem cells from dental pulp, a stem cell separated by the separation method and a population containing the stem cells, differentiation of the stem cells into a hepatocyte lineage and cells differentiated into a hepatocyte lineage.

  Today, regenerative medicine using patients' own cells can avoid the problem of rejection due to histocompatibility mismatch and the bioethical problem due to the use of embryonic stem cells made from fertilized eggs. There is great interest in

  Mesenchymal stem cells are widely used as a cell source for regenerative medicine treatment and research purposes. Mesenchymal stem cells are known to have a high proliferation ability and to differentiate into various cells such as cartilage, muscle, hepatocytes, nerve cells, cardiomyocytes, and vascular endothelial cells. Currently, acquisition of mesenchymal stem cells is performed mainly by acquisition from bone marrow. However, acquisition from the bone marrow is painful because of the surgical invasion of the healthy part.

  In addition, artificial pluripotent stem cells (iPS cells) are expected as cells that can be used in regenerative medicine that can avoid rejection and ethical problems. However, there are a lot of safety issues such as canceration of cells caused by gene transfer.

  Thus, in recent years, knowledge about mesenchymal stem cells present in tooth-related tissues has been reported. Teeth-related tissue is superior in that it is a cell source that can be easily harvested with little surgical invasion. For example, Patent Document 1 discloses acquisition of mesenchymal stem cells by adhesion-separation culture from tooth germs of third molars (wisdom teeth), and induction of the cells into bone, liver, nerves, and the like.

JP 2006-238875 A

  However, in patent document 1, since the tooth germ of the 3rd molar (a wisdom tooth) of bone complete impact is used, (1) The age which can be applied will be restricted (for example, around the late teens). (2) In order to remove a tooth germ, it is necessary to perform a large-scale operation involving a surgical invasion such as jaw bone removal. (3) During extraction surgery, there is a very high possibility of infection with various bacteria (for example, mycoplasma) and viruses (for example, cytomegalovirus) in the oral cavity. (4) It has various problems such as swelling, opening disorder, strong dysfunction after extraction surgery.

  Conventionally, the undifferentiated pluripotent stem cells are separated from the tooth-related tissue cell population by subjecting the cell population to explant culture and selecting the adherent cell group. However, such a method for separating stem cells by adhesion culture may cause a change in properties of the target cells and may be mixed with cells other than the target cells, resulting in variations in the properties of the finally obtained cells. Have a problem that occurs.

  The present inventors have intensively studied to solve the above problems. As a result, we found cell surface antigens useful for isolating mesenchymal stem cells from the pulp of deciduous teeth and third molars (wisdom teeth). Further, the present inventors have found a method capable of preventing contamination of contaminating cells and separating them in a short time by specifically detecting dental pulp-derived mesenchymal stem cells using an antibody against the surface antigen. Surprisingly, the isolated cells have a very high proliferative capacity despite being derived from the pulp (ectodermal), which is a more mature tissue than the tooth germ, and the liver is endodermal. We found that it can be differentiated into a cell lineage. The present invention has been completed based on the above findings.

That is, the gist of the present invention is as follows.
(1) An antibody against at least one of CD117 (c-kit) and CD44 is added to a cell group containing dental pulp-derived mesenchymal stem cells, and a predetermined cell is labeled by a direct labeling method or an indirect labeling method. A method for isolating dental pulp-derived mesenchymal stem cells, which comprises separating using pulp.
(2) The method for separating dental pulp-derived mesenchymal stem cells according to (1), which is performed using at least one of magnetic cell separation (MACS) and flow cytometry (FACS).
(3) The method according to (1) or (2), comprising the following steps (a) to (d): A method for separating dental pulp-derived mesenchymal stem cells.
(A) Step of treating dental pulp with proteolytic enzyme (b) Step of culturing and proliferating the cells obtained in (a) (c) In the cell group cultured in (b), dental pulp-derived mesenchymal stem cells are magnetized Steps (d) and (c) for labeling with beads are added to the separation column, and the labeled cells are collected (4) The pulp is collected from a tooth whose epithelial adhesion is not broken. The method for separating dental pulp-derived mesenchymal stem cells according to any one of (1) to (4).
(5) Dental pulp-derived mesenchymal stem cells that express at least one cell surface antigen of either CD117 or CD44.
(6) Dental pulp-derived mesenchymal stem cells isolated by the separation method according to (1) to (5).
(7) The dental pulp-derived mesenchymal stem cell according to (5) or (6), which can differentiate into an endoderm lineage.
(8) The dental pulp-derived mesenchymal stem cell according to (5) to (7), which can be differentiated into a hepatocyte lineage.
(9) A cell population comprising the dental pulp-derived mesenchymal stem cells according to (5) to (8).
(10) The cell population according to (9), wherein in the cell population, at least 25% expresses cell surface antigen CD117, at least 40% expresses Oct3 / 4, and at least 85% expresses nanog.
(11) A method for subculture of a cell population according to (9) or (10), wherein an antibody against at least one of CD117 and CD44 is added to the cell population at least once every 5 passages, A cell passage method for maintaining a cell line of dental pulp-derived mesenchymal stem cells by labeling cells with a direct labeling method or an indirect labeling method.
(12) A cell line according to (9) or (10), wherein the cell population doubling number is 50 times or more.
(13) A method for inducing differentiation of dental pulp-derived mesenchymal stem cells according to (5) to (8) into a hepatocyte lineage, comprising the following steps (e) and (f): A method for inducing differentiation of mesenchymal stem cells into a hepatocyte lineage.
(E) Step of culturing in DMEM medium containing 2% FBS and 20 ng / ml HGF (f) 2% FBS, 20 ng / ml HGF, 10 ng / ml oncostatin, 10 nM dexamethasone, 50 ng / ml insulin-transferrin-selenium X (ITS- Step 14 of culturing in DMEM medium containing X) Cells of hepatocyte lineage differentiated from dental pulp-derived mesenchymal stem cells according to (5) to (8).
(15) Hepatocyte lineage cells differentiated by the method according to (13).

  By using the method for separating dental pulp-derived mesenchymal stem cells of the present invention, stem cells that can differentiate into endoderm lineage can be obtained from the dental pulp of deciduous teeth and third molars (wisdom tooth). Further, contamination of contaminating cells can be prevented, and a pulp stem cell population with higher purity than before can be obtained.

  This is because while the milk teeth that had been discarded at the time of tooth extraction and the erupted third molar (a wisdom tooth) were used as the source of material, the tooth was collected and separated from the dental pulp of the tooth while reducing the burden of surgical invasion. Since leaf stem cells can be stored and used, it can greatly contribute to the early realization of regenerative medicine avoiding ethical problems, rejection problems, and carcinogenesis.

It is a fluorescence micrograph of dental pulp-derived mesenchymal cells immunostained with an anti-CD117 antibody. It is a fluorescence micrograph of dental pulp-derived mesenchymal cells immunostained with an anti-CD44H antibody. It is a fluorescence micrograph of dental pulp-derived mesenchymal cells immunostained with an anti-Oct3 / 4 antibody. It is a fluorescence micrograph of dental pulp-derived mesenchymal cells immunostained with an anti-Nanog antibody. It is a fluorescence micrograph of dental pulp-derived mesenchymal cells immunostained with an anti-nestin antibody. It is a fluorescence micrograph of dental pulp-derived mesenchymal cells immunostained with anti-cytokeratin 19 antibody. It is a fluorescence micrograph of a dental pulp-derived mesenchymal cell immunostained with an anti-alkaline phosphatase antibody. It is a fluorescence micrograph of dental pulp-derived mesenchymal cells immunostained with an anti-SPARC (ostonectin) antibody. It is a fluorescence micrograph of dental pulp-derived mesenchymal cells immunostained with anti-cytokeratin 18 antibody. FIG. 10 shows the results of flow cytometry using dental pulp-derived mesenchymal cells using anti-Oct3 / 4, Nanog, and CD117 antibodies. It is a light micrograph of dental pulp-derived mesenchymal stem cells induced to differentiate into hepatocyte lineage. It is a fluorescence micrograph of dental pulp-derived mesenchymal cells immunostained with an anti-albumin antibody. It is a fluorescence micrograph of dental pulp-derived mesenchymal cells immunostained with an anti-IGF-1 antibody. It is a figure which shows the urea production amount of the dental pulp origin mesenchymal stem cell which induced hepatocyte lineage differentiation.

In the present invention, the dental pulp is a connective tissue rich in blood vessels and nerves found in the core of the tooth, and means a tooth papilla grown in a tooth germ. In the present invention, pulps derived from mammals, for example, primates such as humans can be used.
1. Method for separating dental pulp-derived mesenchymal stem cells of the present invention

  In the method for separating dental pulp-derived mesenchymal stem cells of the present invention, an antibody against at least one of CD117 (c-kit) and CD44 is added to a cell group containing dental pulp-derived mesenchymal stem cells, and predetermined cells are directly labeled. It is characterized by labeling by the method or indirect labeling method, and separating using the label.

  The tooth as a dental pulp supply source in the present invention may be any tooth as long as it is in the oral cavity and is not invaded by bacteria, that is, the epithelial adhesion due to so-called hemidesmosome binding is not broken. Teeth erupted on the gingiva may be mentioned, and specific examples include deciduous teeth (incisors (incisors), canines, molars) or third molars (wisdom teeth). Further, the root resorption stage does not matter. In addition, the tooth | gear used as a dental pulp supply source may be once frozen and preserve | saved after extraction, and may be thawed | decompressed as needed.

  The dental pulp used in the present invention can be collected by methods well known to those skilled in the art. For example, in the case of extracted deciduous teeth, the roots are almost completely absorbed and the crown pulp is exposed in the alveolar bone. Therefore, immediately after tooth extraction, it is extracted from this exposed portion with an excavator or a pulp removal cleanser. In the case of the third molar, a groove is made around the tooth neck with a dental cutting bar, a dental spatula is inserted and divided, and the root and crown pulp is collected according to the method of the deciduous tooth.

  The dental pulp-derived cells to be subjected to separation in the present invention can be obtained by treating the collected dental pulp with a proteolytic enzyme in an appropriate buffer or culture solution. Examples of proteolytic enzymes include collagenase, trypsin, hyaluronidase, elastase, pronase, and dispase, and it is more preferable to use type I collagen.

In addition, before proceeding to the separation step, the obtained cell suspension is preferably made into a single cell suspension by removing cell clumps using a filter or the like. In addition, it is preferable to grow the said cell to the quantity (for example, 1 * 10 < ⁸ >) required for a isolation | separation step.

The dental pulp cells to be separated and the dental pulp-derived mesenchymal stem cells separated by the method of the present invention can be cultured using a normal serum-containing medium or serum-free medium used for animal cell culture. Under the conditions. For example, using DMEM (Dulbecco's Modified Eagle's Medium) medium containing 20% FBS (fetal bovine serum), it can be performed by static culture in a 37 ° C., 5% CO ₂ incubator.

  Separation of dental pulp-derived mesenchymal stem cells of the present invention is performed by labeling cells using any antibody that recognizes the antigen expressed on the surface of dental pulp-derived mesenchymal stem cells, and separating using the label. To do.

  Here, the antibody that recognizes the antigen expressed on the surface of dental pulp-derived mesenchymal stem cells may be any antibody that can recognize mesenchymal stem cells, but recognizes the CD44 antigen. The antibody that recognizes the CD117 (c-kit) antigen is more preferable.

  As a method for labeling cells, either a direct labeling method or an indirect labeling method may be used. Here, the direct labeling method refers to a method in which the detection antibody (primary antibody) itself is labeled and performs cell labeling by a single antigen-antibody reaction, and the indirect labeling method does not label the detection antibody (primary antibody). In addition, it refers to a method of labeling a cell by labeling another antibody (secondary antibody) that detects the detection antibody. For example, indirect labeling is performed by labeling cells with a primary antibody, either fluorescent dye-labeled, biotin-labeled or unlabeled, and then using either an anti-fluorescent dye antibody, anti-biotin antibody, streptavidin or anti-IgG antibody. It can be carried out.

As the separation means, there can be used magnetic separation method (MACS) for labeling and separating cells with an antibody to which magnetic beads are bound, and flow cytometry (FACS) for labeling and separating cells with a fluorescent dye. Here, the magnetic separation method (MACS) can be performed on a small scale, and since there is little physical stress on the cells during the separation, cells with high recovery and viability can be easily obtained in a short time. More preferable as the separation means of the present invention. Since magnetic beads are extremely small in size, they are biologically degraded in the culture after separation, and do not affect the function and survival rate of subsequent cells.
2. Dental pulp-derived mesenchymal stem cell of the present invention, cell population containing the cell, and cell line

  The dental pulp-derived mesenchymal stem cells, the cell population containing the cells and the cell line of the present invention are the dental pulp-derived mesenchymal stem cells isolated by the method of the present invention, the cell population containing the cells and the cell line, And

The cell population containing dental pulp-derived mesenchymal stem cells obtained by the method of the present invention has a very high proliferation ability, and a cell line can be established by performing subculture under normal culture conditions. For example, using a DMEM (Dulbecco's Modified Eagle's Medium) medium containing 20% FBS (fetal bovine serum) in a 25 cm ² flask until 80% confluent, 0.05% trypsin / 0.53 mM Cell lines can be established by subculturing by detaching cells using EDTA PBS and reseeding at a 1: 3 split ratio.

  In the passage of the cell population, an antibody against a molecule present in the dental pulp-derived mesenchymal stem cell is added to the cell population at least once every 5 passages, and a predetermined cell is directly or indirectly labeled. The cell line of dental pulp-derived mesenchymal stem cells may be maintained by labeling. Examples of the molecule include, but are not limited to, CD117 or CD44. Two or more kinds of antibodies may be used in combination.

  The cell line containing dental pulp-derived mesenchymal stem cells of the present invention preferably has a cell population doubling number of 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more. It is preferable.

  It can be confirmed that the dental pulp-derived mesenchymal stem cells, cell populations and cell lines containing the cells of the present invention show properties close to mesenchymal stem cells by immunohistochemical staining and flow cytometry. As the marker, those known to those skilled in the art as markers for undifferentiated stem cells, pluripotent stem cells, and mesenchymal stem cells can be used. For example, CD117, CD44H, Oct3 / 4, Nanog, nestin, cytokeratin 19, Alkaline phosphatase (ALP), SPARC (ostonectin), CD24, CD34, CD41, CD45, CD133, Lin (Lineage marker), Sca-1 and Flt3 / Flk2 can be used. In addition, a differentiation marker can be used in combination with an undifferentiation marker, for example, cytokeratin 18 can be used.

  The pulp-derived mesenchymal stem cell of the present invention and a cell line containing the cell are positive for CD117, CD44H, Oct3 / 4, Nanog, nestin, cytokeratin 19, alkaline phosphatase (ALP) and SPARC (ostonectin) It is preferably keratin 18 negative.

  The cell population containing dental pulp-derived mesenchymal stem cells of the present invention preferably has 40% or more of Oct3 / 4 positive cells, preferably 50% or more, and preferably 65% or more. In addition, Nanog positive cells are preferably 85% or more, preferably 95% or more, and preferably 99% or more. Furthermore, CD117 positive cells are preferably 25% or more, preferably 35% or more, and preferably 45% or more.

In addition, the pulp-derived mesenchymal stem cell of the present invention may be one into which an arbitrary gene has been introduced. For example, iPS cells that are said to have properties close to totipotency may be produced from the dental pulp-derived mesenchymal stem cells of the present invention. iPS cells can be prepared by introducing genes that promote transition to a dedifferentiated state, such as Sox2, Oct3 / 4, KLF4, and c-myc.
3. Method for inducing differentiation of dental pulp-derived mesenchymal stem cells of the present invention into hepatocyte lineage

  The dental pulp-derived mesenchymal stem cells of the present invention may be differentiated into any of the endoderm lineage, mesoderm lineage, and ectoderm lineage. Examples thereof include various cells such as bone cells, chondrocytes, hepatocytes, pancreatic cells, fat cells, nerve cells, cardiomyocytes, vascular endothelial cells, and tooth-related cells and tissues. Examples of the tooth-related cells and tissues include odontoblasts, periodontium, dentin, cementum, enamel, periodontal tissue, connective tissue, and the like. Differentiation into various cells and tissues can be performed by methods well known to those skilled in the art.

  For example, the dental pulp-derived mesenchymal stem cells of the present invention can be differentiated into an endoderm hepatocyte lineage. First, the cells are cultured for about 5 days in a medium containing hepatocyte growth factor (HGF) and fetal bovine serum (FBS). The HGF concentration is preferably 20 ng / ml, the FBS concentration is 2%, and the basic medium is preferably DMEM (Dulbecco's Modified Eagle's Medium), but is not limited thereto. Next, the cells are cultured for about 15 days in a medium containing dexamethasone, insulin-transferrin-selenium X (ITS-X), and oncostatin. Preferably, the dexamethasone concentration is 10 nM, the insulin-transferrin-selenium X (ITS-X) concentration is 50 ng / ml, the oncostatin concentration is 10 ng / ml, and the basic medium is IMDM (Iscove's Modified Dulbecco's Medium). It is not limited to these.

  Differentiation into the endoderm or hepatocyte lineage of the present invention can be confirmed by cell morphology. For example, deciduous dental pulp-derived mesenchymal stem cells have a fibroblast-like spindle-like shape, whereas cells after induction of differentiation into a hepatocyte lineage have a parenchymal-like polygonal form.

  The degree of differentiation can be confirmed by immunohistochemical staining using a differentiation marker or flow cytometry. In cells differentiated into the hepatocyte lineage, for example, α-fetoprotein, albumin, and insulin growth factor (IGF-1) can be used as markers.

  The cells of the present invention can be stored frozen. That is, dental pulp-derived mesenchymal stem cells isolated from dental pulp and / or cells differentiated from the cells into hepatocyte lineage (including primary culture, subcultured cells, and cell line) are placed in a cell cryopreservation solution, and -80 ° C. It can be stored frozen in the following freezer. The cell cryopreservation solution and the cell cryopreservation method can be performed by methods well known to those skilled in the art. The cell cryopreservation solution is preferably composed of 10% DMSO, 30% FBS, and 60% DMEM, but is not limited thereto.

  In addition, the cryopreserved cells can be thawed and washed by a method well known to those skilled in the art when the cells are needed, and the cells can be proliferated or differentiated into the target cells as necessary. Thereby, for example, it becomes possible to quickly respond to the needs of patients who need tissue repair.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

[Sample preparation]
Informed consent was obtained from human deciduous teeth and third molars that were no longer needed, and the pulp was collected using a barbed broach. The collected dental pulp was placed in DMEM (20% FCS), transferred to a 15 ml conical centrifuge tube, and then 1 ml of 3 mg / ml collagenase type I (Waco Pure Chemical Industries Ltd. # 03-1760) was added to cut the tissue. . After tissue cutting (maximum 10 min), centrifuge at 1000 × g for 5 min, remove supernatant, and cell pellet with 2 ml DMEM (# Invitrogen 12800-017) supplemented with 20% FBS (Invitrogen, Sigma, HyClone) It was suspended in. Thereafter, the suspension was passed through a filter (Miltenyi Biotech GmbH # 130-041-407) having a pore size of 30 μm to remove cell clumps.

The obtained single cell suspension was seeded in a 25 cm ² flask using DMEM (20% FBS) medium, and cultured at 37 ° C. under 5% CO ₂ conditions. Every 2-3 days, the medium was changed with DMEM (10% FBS), and the cells were cultured until they became 80% confluent. After becoming 80% confluent, the medium was removed using a pipette and washed with 5 ml PBS (Invitrogen # 14200) solution for 3 minutes. Cells were then incubated for 5-7 minutes in 0.05% trypsin / 0.53 mM EDTA (Invitrogen # 25300-054) PBS. The cell suspension was collected in a 15 ml conical centrifuge tube (with tissue culture solution), centrifuged at 1000 × g for 5 minutes, the supernatant was removed, and the cell pellet was suspended in 3 ml of DMEM (20% FBS). .

[Separation of CD117 positive cells by magnetic cell separation (MACS)]
The following operation was quickly performed using the cooled solution while keeping the cells in a cooled state.

After measuring the cell number of the cells prepared in Example 1, the cell suspension was centrifuged at 300 × g for 10 minutes, and the supernatant was completely removed. The cell pellet was suspended in 300 μl buffer per 1 × 10 ⁸ cells. As a buffer, MACS BSA Stock Solution (Miltenyi Biotech GmbH # 130-091-376) and autoMACS (registered trademark) Rising Solution (Miltenyi Biotech GmbH # 130-091-222) were mixed at 1:20. 5% BSA, 2 mM EDTA in PBS) was used after degassing. Further, the cell suspension was mixed with the addition of 1 × 10 ⁸ pieces of cells per 100μl FcR Blocking Reagent, 1 × 10 ⁸ cells per 100μl CD117 MicroBeads (Miltenyi Biotech # 130-091-332 ) Incubated for 15 minutes at 4 ° C. Thereafter, 1 ml of buffer per 1 × 10 ⁸ cells was added to wash the cells, centrifuged at 300 × g for 10 minutes, the supernatant was completely removed, and the cell pellet was 500 μl per 1 × 10 ⁸ cells. In the buffer.

  A column (MS: Miltenyi Biotech GmbH # 130-042-201) was placed on a separator (Mini MACS: Miltenyi Biotech GmbH), and the column was rinsed with 500 μl of buffer, and then the cell suspension was applied to the column. The column was washed 3 times with 500 μl of buffer and the eluted unlabeled cell fraction was collected. After removing the column from the separator and placing it on the tube, 1 ml of buffer was applied to the column, and the magnetically labeled cell fraction was eluted with a plunger.

[Establishment of cell lines containing dental pulp-derived mesenchymal stem cells]
The obtained CD117 positive cells were suspended in DMEM (20% FBS) medium, seeded in a 25 cm ² flask, and cultured for 1 day under conditions of 37 ° C. and 5% CO ₂ . Every 2-3 days, the medium was changed with DMEM (10% FBS), and the cells were cultured until they became 80% confluent. After becoming 80% confluent, the medium was removed using a pipette and washed with 5 ml PBS solution for 3 minutes. Cells were then incubated for 5-7 minutes in 0.05% trypsin / 0.53 mM EDTA (Invitrogen # 25300-054) PBS. The cell suspension was collected in a 15 ml conical centrifuge tube (with tissue culture solution), centrifuged at 1000 × g for 5 minutes, the supernatant was removed, and the cell pellet was suspended in 3 ml of DMEM (20% FBS). . Then, cells containing CD117-positive dental pulp-derived mesenchymal stem cells that are subcultured at a split ratio of 1: 3 using DMEM (20% FBS) and 25 cm ² flasks and have a cell population doubling number of 50 or more. A line was established.

[Marker expression analysis of dental pulp-derived mesenchymal cells]
The expression of the marker protein of the human dental pulp-derived mesenchymal cells obtained in Examples 1 to 3 was analyzed by immunostaining and flow cytometry.

  The results of immunostaining for CD117, CD44H, Oct3 / 4, Nanog, nestin and cytokeratin 19 are shown in FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. The results of immunostaining for alkaline phosphatase (ALP) and SPARC (ostonectin) are shown in FIGS. 7 and 8, respectively. The results of immunostaining for cytokeratin 18 are shown in FIG.

  As shown in FIG. 1 to FIG. 6, it is confirmed that CD117, CD44H, Oct3 / 4, Nanog, nestin, and cytokeratin 19 that are markers of pluripotent stem cells are expressed in human dental pulp-derived mesenchymal cells. did. Moreover, as shown in FIG.7 and FIG.8, it confirmed that the alkaline phosphatase (ALP) and SPARC (ostonectin) which are the markers of an undifferentiated cell are expressing in the cell. On the other hand, as shown in FIG. 9, it was confirmed that cytokeratin 18, which is a differentiation marker for epithelial or endoderm cells, was not expressed in the same cells.

  FIG. 10 shows the results of flow cytometry for unlabeled cells (control), Oct3 / 4, Nanog, and CD117. In the obtained cell population, it was confirmed that Oct3 / 4 positive cells were 65%, Nanog positive cells were 99%, and CD117 positive cells were 45%.

  From these results, it was confirmed that the human dental pulp-derived mesenchymal cells obtained in Examples 1 to 3 exhibited the properties as pluripotent stem cells and maintained the undifferentiated state. .

[Induction of differentiation of dental pulp-derived mesenchymal stem cells into hepatocyte lineage]
Using human dental pulp-derived mesenchymal stem cells obtained in Examples 1 to 3, differentiation induction into a hepatocyte lineage was performed. Differentiation induction was first cultured for 5 days in DMEM medium containing 20 ng / ml HGF (R & D Systems Inc. # 294-HG) and 2% FBS, and then 10 nM dexamethasone (Waco Pure Chemical Industries Ltd. # 047). 18863), IMDM (#Invitrogen 12200-036) medium containing 50 ng / ml insulin-transferrin-selenium X (Invitrogen ITS-X # 51500) and 10 ng / ml oncostatin (R & D Systems Inc. # 295-OM) For 15 days.

  FIG. 11 shows cell photographs before and after induction of differentiation of the dental pulp derived from deciduous teeth into the hepatocyte lineage. It can be seen that the cells before differentiation induction are in the form of spindle-like fibroblasts, whereas the cells after differentiation induction are in the form of parenchymal cell-like polygons.

[Analysis of differentiation marker expression in cells induced to differentiate into hepatic lineage]
The expression of differentiation marker proteins of the dental pulp-derived mesenchymal stem cells of the deciduous teeth and the dental pulp-derived mesenchymal stem cells of the third molar (parental wisdom tooth) differentiated into the hepatocyte lineage by the method of Example 5 was analyzed by immunostaining.

  The results of immunostaining using anti-albumin antibody (R & D Systems Inc., Minneapolis, USA) and anti-insulin-like growth factor (IGF-1) antibody (Raybiotech, Norcross, USA) are shown in FIGS. 12 and 13, respectively. As shown in FIG. 12, it was confirmed that albumin, which is a protein produced by hepatocytes, was expressed in both dental pulp-derived mesenchymal stem cells of deciduous teeth and third molars. In addition, as shown in FIG. 13, in both the dental pulp-derived mesenchymal stem cells of deciduous teeth and third molars, some cells are insulin-like growth factor-1 (IGF-), which is a protein mainly produced by hepatocytes. It was confirmed that 1) was expressed. It can be seen that the dental pulp-derived mesenchymal stem cells of the deciduous teeth and the dental pulp-derived mesenchymal stem cells of the third molar are differentiated into cells of the hepatic cell lineage.

[Analysis of urea production in cells induced to differentiate into hepatic lineage]
The amount of urea produced by the dental pulp-derived mesenchymal stem cells of the deciduous teeth and the dental pulp-derived mesenchymal stem cells of the third molar (parental wisdom tooth) differentiated into the hepatocyte lineage by the method of Example 5 was analyzed by ELISA.

  FIG. 14 shows the results of analysis using the Quantum Chure Urea Assay Kit (BioAssay Systems, USA) on the dental pulp derived mesenchymal stem cells of the deciduous teeth and the dental pulp derived mesenchymal stem cells of the third molar. Shown in It was confirmed that the urea concentration in the culture broth increased by 4 to 15% compared to the control medium. It can be seen that the dental pulp-derived mesenchymal stem cells of the deciduous teeth and the dental pulp-derived mesenchymal stem cells of the third molar are differentiated into cells of the hepatic cell lineage.

[Cell cryopreservation]
Media was removed, cells were washed with 5 ml PBS for 3 minutes, and cells were incubated in 0.05% trypsin / 0.53 mM EDTA (Invitrogen # 25300-054) PBS for 5-7 minutes. Thereafter, the cell suspension was collected in a 15 ml conical centrifuge tube (containing a tissue culture solution), centrifuged at 1600 rpm for 5 minutes, the supernatant was removed, and the cell pellet was recovered in 1500 μl of freezing medium (10% DMSO, 30% FBS). , 60% DMEM). 500 μl of the cell suspension was dispensed into cryovials, placed at −80 ° C. for 24 hours, and stored for a long time in liquid nitrogen.

  Since the present invention can be carried out especially using deciduous teeth and erupted wisdom dental pulps, there are problems of surgical invasion, ethical problems and rejection problems associated with cell source collection, and regenerative medicine avoiding carcinogenesis. It can greatly contribute to early realization.

Claims (15)

An antibody against at least one of CD117 (c-kit) and CD44 is added to a cell group containing dental pulp-derived mesenchymal stem cells, and a predetermined cell is labeled by a direct labeling method or an indirect labeling method, and the label is used. And separating the dental pulp-derived mesenchymal stem cells. The method for separating dental pulp-derived mesenchymal stem cells according to claim 1, wherein the method is performed using at least one of magnetic cell separation (MACS) and flow cytometry (FACS). The method according to claim 1 or 2, comprising the following steps (a) to (d): A method for separating dental pulp-derived mesenchymal stem cells.
(A) Step of treating dental pulp with proteolytic enzyme (b) Step of culturing and proliferating the cells obtained in (a) (c) In the cell group cultured in (b), dental pulp-derived mesenchymal stem cells are magnetized Step of labeling with beads (d) Step of adding the cells after the steps of (c) to the separation column and collecting the labeled cells 5. The method for separating pulp-derived mesenchymal stem cells according to claim 1, wherein the pulp is collected from a tooth whose epithelial adhesion is not broken. Dental pulp-derived mesenchymal stem cells that express at least one cell surface antigen of either CD117 or CD44. Dental pulp-derived mesenchymal stem cells isolated by the separation method according to claim 1. The pulp-derived mesenchymal stem cell according to claim 5 or 6, which can differentiate into an endoderm lineage. The dental pulp-derived mesenchymal stem cell according to claim 5, which can differentiate into a hepatocyte lineage. A cell population comprising the dental pulp-derived mesenchymal stem cell according to claim 5. 10. The cell population of claim 9, wherein in said cell population, at least 25% express cell surface antigen CD117, at least 40% express Oct3 / 4, and at least 85% express nanog. The method for subculturing a cell population according to claim 9 or 10, wherein an antibody against at least one of CD117 or CD44 is added to the cell population at least once every 5 times, and a predetermined cell is directly labeled. Or the subculture method of the cell which maintains the cell line of a dental pulp origin mesenchymal stem cell by labeling by the indirect labeling method. The cell population according to claim 9 or 10, wherein the cell population doubling number is 50 times or more. A method for inducing differentiation of dental pulp-derived mesenchymal stem cells according to claim 5 to hepatocyte lineage, comprising the following steps (e) and (f): A method for inducing differentiation into a hepatocyte lineage.
(E) Step of culturing in DMEM medium containing 2% FBS and 20 ng / ml HGF (f) 2% FBS, 20 ng / ml HGF, 10 ng / ml oncostatin, 10 nM dexamethasone, 50 ng / ml insulin-transferrin-selenium X (ITS- Step of culturing in DMEM medium containing X) A cell of the hepatocyte lineage differentiated from the dental pulp-derived mesenchymal stem cell according to claims 5 to 8. A cell of the hepatocyte lineage differentiated by the method of claim 13.