JP2004201612A - Undifferentiated multipotential cell and method for preparing related tissue or tooth by using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an noninvasively and readily available undifferentiated multipotential cell and a transplanted material having high utility value using the cell. <P>SOLUTION: The undifferentiated multipotential cell is obtained from a dental pulp cell or a dental sac cell. The related tissue or the tooth is prepared by mixing the undifferentiated multipotential cell with a osteoblast inducer and β-TCP. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】本発明に係る歯髄細胞及び歯嚢細胞を示す模式図である。
【図２】本発明の実施例にかかる歯髄細胞等のＤＳＰＰ活性を説明するグラフである。
【図３】本発明の実施例にかかる歯髄細胞等のＤＭＰ１活性を説明するグラフである。
【図４】本発明の実施例にかかる歯髄細胞等のＤＳＰ活性を説明するグラフである。
【図５】（Ａ）は、本発明の実施例にかかる歯髄細胞等のＤＭＰ１活性を説明するグラフ、（Ｂ）は、（Ａ）と同一の細胞のＡＬＰ活性を説明するグラフである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an undifferentiated pluripotent cell and a method for producing a related tissue or tooth using the same, and particularly to a highly undifferentiated pluripotent cell which can be used not only in the field of dentistry and this undifferentiated pluripotent cell. The present invention relates to a related tissue or tooth preparation method used.
[0002]
[Prior art]
In the current transplantation technology, self or another person's tissue is used for repairing a self tissue lost due to an accident or the like. In particular, it is generally well known that it is preferable to use a tissue with a matching histocompatibility antigen to avoid problems such as rejection. When the number of cells to be collected is insufficient, for example, cultured cells are also used. However, the method of collecting and culturing cells of the same kind has a problem that it cannot cope with a case where cells to be cultured cannot be collected in the first place or a case where appropriate cells cannot be collected from other sites. In many cases, the situation of receiving a transplant is very urgent, and in such a case, it takes too much time to wait for the supply of the cells to be transplanted. In order to solve this, it has been proposed to collect stem cells from unnecessary tissues in advance, or to use precursor cells capable of inducing differentiation into a plurality of cell types, ie, stem cells.
[0003]
It is known that embryonic stem cells (ES cells) among stem cells can be differentiated into all kinds of tissues and have high proliferation ability. However, since there are many ethical problems in handling embryonic stem cells, the use of stem cells present in other organs instead of embryonic stem cells has been proposed.
In particular, mesenchymal stem cells, which have been confirmed to differentiate into cells such as bone, cartilage, heart, nerve, and tendon, have attracted attention because they cover a range of applicable organs in high demand. Such mesenchymal stem cells include bone marrow (see Non-Patent Documents 1 and 2), peripheral blood (see Non-Patent Document 3), umbilical cord blood (see Non-Patent Document 4), adipocytes (see Non-Patent Document 5), and the like. Or their presence in these tissues has been suggested.
[0004]
[Non-patent document 1]
Mark F. Pittenger, et.al., Science, (1999) vol.284, pp143-147
[Non-patent document 2]
Katia Mareschei, et.al., Haematologica (2001) vol.6, pp1099-1100
[Non-Patent Document 3]
Sergei A. Kuznetsov, et al., The journal of Cell Biology, (2001) vol.153, pp1133-1139
[Non-patent document 4]
Alejandro Erices, et al., British Journal of Haematology (2000) vol.109, pp235-242
[Non-Patent Document 5]
Patricia A. Zuk, et al., Tissue Engineering (2001) vol.7 (2), pp211-228
[0005]
[Problems to be solved by the invention]
However, isolation methods from any of these tissues are invasive and painful. Further, it is said that stem cells obtained from cord blood have many blood stem cells and few mesenchymal stem cells. On the other hand, mesenchymal stem cells derived from bone marrow must rely on bone marrow from a bone marrow bank, but at present, the number of bone marrow registered in the bone marrow bank is not sufficient. Moreover, methods for isolating mesenchymal stem cells from any tissue are not complete.
Thus, although the existence of mesenchymal stem cells in multiple organs has been suggested, it is not easy to obtain such undifferentiated pluripotent cells, and it is recognized that they are highly useful cells. Despite this, it is currently not meeting its high and urgent needs.
Therefore, an object of the present invention is to provide an undifferentiated pluripotent cell which can be obtained non-invasively and easily, and a highly useful transplant using the same.
[0006]
[Means for Solving the Problems]
The undifferentiated pluripotent cell of the present invention is characterized by being an undifferentiated pluripotent cell derived from a tooth-related cell.
The related tissue or tooth preparation method of the present invention is characterized in that undifferentiated pluripotent cells are obtained from tooth-related cells, and a related tissue or tooth is prepared from the obtained undifferentiated pluripotent cells.
In the above invention, the tooth-related cell is preferably at least one of a dental pulp cell and a dental pulp cell.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
The undifferentiated pluripotent cell of the present invention is derived from a tooth-related cell. That is, the present invention is based on the finding that tooth-related cells include undifferentiated pluripotent cells capable of differentiating into various types of cells. Unlike the bone marrow and the like, the tooth-related cells can use unnecessary teeth such as wisdom teeth and can be easily obtained. Therefore, according to the present invention, useful undifferentiated pluripotent cells can be obtained by a simpler route.
[0008]
The tooth-related cell in the present invention refers to a group of cells constituting the tooth in the oral cavity of a mammal, and a group of cells attached to or around the tooth, and is preferably at least a dental pulp cell and a dental pulp cell. On the other hand.
As shown in FIG. 1, the tooth before eruption (tooth germ) is buried in the oral mucosal tissue and is wrapped by a tooth capsule.
Here, the dental pulp cells are a group of connective tissue cells rich in blood vessels and nerves found in the core of the tooth. Dental sac cells are a group of cells in the alveolar process that constitute a sac that encloses the tooth before eruption. In the present invention, the dental pulp cells and the dental pulp cells mean the cell groups specified at such anatomical positions in the living body, respectively.
[0009]
These cells can be collected by methods known in the art. In particular, it is preferably derived from a tooth obtained by tooth extraction accompanying tooth treatment, or a tooth separated from the gum together with the tooth at the time of tooth extraction. In many cases, tooth extraction is performed due to an abnormality / problem in the tooth, and thus, the tooth-related cells available with the tooth extraction retain the characteristics of the cells. For this reason, it is advantageous to use such tooth-related cells.
[0010]
In particular, so-called "wisdom teeth" are often unnecessary and tooth extraction is often performed, and tooth extraction without wisdom teeth may be performed when not yet erupted. For this reason, it is particularly preferable to collect dental capsule cells surrounding the tooth. By adopting such a method for obtaining cells, tooth-related cells that have been conventionally treated as waste can be effectively used, and the cells can be newly obtained without pain for the purpose of obtaining cells. Of cells can be obtained.
[0011]
If the collected tooth-related cells contain, for example, connective tissue or debris, they can be removed using an appropriate enzyme, for example, dispase, trypsin, or a nylon mesh.
[0012]
The undifferentiated pluripotent cells of the present invention can be easily isolated by subjecting the tooth-related cell population collected as described above to an explant culture to select a group of adherent cells. can do. Media used herein include those known in the art of cell culture, such as Dulbecco's Modified Eagle's Minimal Medium (DMEM), including 10% FBS. At this time, the culture conditions are preferably 5% CO ₂ , room temperature 37 ° C., and humidity 90%. In addition, these culture conditions and culture medium are similarly applied to culture for increasing the number of collected undifferentiated pluripotent cells.
[0013]
The undifferentiated pluripotent cells of the present invention are adherent cells capable of differentiating into various types of cells. These cells show properties close to those of undifferentiated mesenchymal stem cells, and have the same surface markers as mesenchymal stem cells, such as CD105, CD106, CD29, and CD44, and negatively express CD14, CD34, and CD45. it is conceivable that.
The undifferentiated pluripotent cell of the present invention preferably has an ability to induce odontoblasts having dentin-forming ability. In particular, undifferentiated pluripotent cells derived from dental pulp cells or dental pulp cells have the ability to induce odontoblasts. This ability to induce odontoblasts can be confirmed by a method known in the art. That is, by adding an inducer known to induce differentiation into a specific cell to a medium and culturing it, the cell can be differentiated into a specific cell. The induction of differentiation into a cell group having a characteristic morphology can be confirmed by the morphology after differentiation.
[0014]
In particular, the ability to induce odontoblasts can be confirmed by adding an osteoblast-inducing factor. Examples of osteoblast-inducing factors include dexamethasone, ascorbic acid, β-glycerophosphate, and the like.
[0015]
The undifferentiated pluripotent cell of the present invention can be differentiated based on its function and used to supply various types of cells. Compared to directly collecting target cells from individual tissues or obtaining stem cells from bone marrow or the like in order to obtain various types of cells, by using the undifferentiated pluripotent cells of the present invention, Cells can be obtained easily and in large quantities.
[0016]
In particular, the present invention includes using the above undifferentiated pluripotent cells to produce related tissues or teeth from the undifferentiated pluripotent cells.
Here, the term “related tissue or tooth” refers to a tissue related to undifferentiated pluripotent cells, and the related tissue includes bone, cartilage, nerve, and the like. Tissues existing around the teeth, such as periodontal tissue, dentin, cementum, enamel, bone, periodontal ligament tissue, connective tissue and the like are included. The production of the “related tissue or tooth” includes not only the production of either one but also the production of both of them.
Producing a related tissue or tooth from undifferentiated pluripotent cells involves inducing differentiation into a target tissue using a known inducer, for example, using an osteoblast inducer to increase dentin-forming ability. Stimulation is included.
Preferably, mixing the undifferentiated pluripotent cells, the osteoblast-inducing factor described above, and a carrier matrix is included. Examples of the carrier matrix include ceramics such as hydroxyapatite, phosphate compounds such as β-TCP (β-tricalcium phosphate), platelet-rich plasma (PRP), absorbable collagen, and fibrin glue. As β-TCP, those having a porosity of 90% and a pore diameter of 200 to 400 μm are preferable.
[0017]
The mixing amount of the osteoblast-inducing factor and the carrier matrix for the undifferentiated pluripotent cells may be the amount usually used for this purpose and is known to those skilled in the art. Preferably, when the number of undifferentiated pluripotent cells is 10 ⁶ cells / ml, the osteoblast-inducing factor is 10 ⁻⁵ M to 10 ⁻¹² M dexamethasone, and ⁵ mM to 25 mM β-glycerophos. Fate and 0.01 mM to 1 mM ascorbic acid are each selected. It is particularly preferable to use ^10-8 M dexamethasone, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid, respectively.
Mixing is carried out by immersing the carrier in a cell suspension at the above concentration and culturing the carrier. PRP is mixed with 10 ⁷ cells / ml with 10% CaCl ₂ / thrombin in a mixing ratio of 1: 1 to 10: 1, preferably 6: 1.
[0018]
The tooth obtained by the present invention includes a tooth formed from dentin or a part thereof, and can be used as a tooth or a tooth fragment for various dental treatments. Thus, the present invention can be applied to the treatment of a tooth defect which has been conventionally performed using an inorganic substance, and can be restored to a state close to a living body.
In addition, a large amount of a tooth or a tooth fragment, a tissue around a tooth, or a tissue related to undifferentiated pluripotent cells obtained in accordance with the present invention can be prepared in advance in a large amount and prepared when necessary. Thereby, various kinds of tissues can be easily provided.
Furthermore, it is also conceivable that the related tissue or tooth according to the present invention has low immunogenicity, in which case it can be widely used independently of home and other.
[0019]
【Example】
1. Recovery of undifferentiated pluripotent cells and induction of differentiation [Sample preparation]
Human third molar tooth germs that became unnecessary and were extracted were obtained after obtaining informed consent. After collection, the dental pulp tissue and dental pulp tissue were cut off and collected from the tooth germ as soon as possible (see FIG. 1). Oral mucosal cells were collected from mucosal tissues around tooth germs.
Each tissue was subjected to explant culture in a 75 cm ² flask. The medium used was Gibco's Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 4 mM glutamine and 0.05 U / ml penicillin / streptomycin. The culture was performed at 37 ° C. under 5% CO _{2 for} about 30 days to obtain a sample. The medium was changed every 2-3 days, and the cells were subcultured as usual in the art according to the cell density. The resulting cells were identified by their adherence and morphology.
[0020]
[Collection of RNA]
Cells obtained by explant culture were each induced to differentiate into odontoblasts using a medium to which an osteoblast-inducing factor was added.
The seeding density of the cells was 3.1 × 10 ³ cells / cm ² in a dish having a diameter of 10 cm. Using medium, 20% fetal bovine serum (FBS), 4 mM glutamine and 0.05 U / ml penicillin, supplemented with streptomycin 0.05 [mu] g / ml, further, as osteoblast inducers, ¹⁰⁶ cells / ml In each case, a DMEM medium supplemented with 10 ⁻⁸ M dexamethasone, 0.05 mM ascorbic acid diphosphate, and 10 mM β-glycerophosphate was used.
RNA was collected on each cell on days 3, 6, 9, 15, 21, and 27 using an RNA collection kit (RNeasy) manufactured by Qiagen. Harvesting followed a known protocol. The collected RNA was stored frozen at -140 ° C using a cell banker.
[0021]
[Real-time RT-PCR]
The real-time RT-PCR was measured using AB1 Prism 7000 Sequence Detection System (AB1 Prisum 7000 Sequence detection system) manufactured by Applied Biosystems. The following were prepared and used as primers and Taqman probes used in the sequence. Dentin sialoprotein (DSP), dentin sialophosphoprotein (DSPP), dentin phosphoprotein (DPP) and dentin matrix protein 1 (DMP1) are all known to be markers expressed in odontoblasts during the differentiation and maturation process. ing.
[0022]
DSPP:
DSPP-163F (GCCATTCCAGTTCCTCAAAGC: SEQ ID NO: 1)
DSPP-307R (CATGCACCAGGACACCACTT: SEQ ID NO: 2)
DSPP-283Probe (TGATGGTTCCACTGGCATTTAACTCATCC: SEQ ID NO: 3)
DMPl
DMP1-105F (GATCAGCATCCTGCTCATGTTC: SEQ ID NO: 4)
DMP1-214R (GAGCCAAATGACCTTCCATT: SEQ ID NO: 5)
DMP1-140Tprobe (CCTGTGCTCTCCCAGTAACCAGGTATCAAA: SEQ ID NO: 6)
DSP:
DSP-3083F (GTGAAATATCTGGCAAACGAGACA: SEQ ID NO: 7)
DSP-3204R (CAGAGTTTCCAGTCCTGAGGTGTA: SEQ ID NO: 8)
DSP-3127Tprobe (ATTGCTGAACTTTGCGCCAATTCA: SEQ ID NO: 9)
DPP:
DPP-39F (TGATGCTAATTCAGAAAGTGACAATAAC: SEQ ID NO: 10)
DPP-154R (CATCATCTTCTGCTCCTTTTGAGTC: SEQ ID NO: 11)
DPP-103Tprobe (CATCAGAGTTATAAGAAGCATCTCCTCGGC: SEQ ID NO: 12)
[0023]
The results are shown as graphs in FIG. 2 (DSPP), FIG. 3 (DMP1) and FIG. 4 (DSP), respectively. Each graph is a numerical graph of the measurement results of real-time RT-PCR and relatively graphed. X indicates undifferentiated pluripotent cells derived from dental pulp cells, and ◇ indicates undifferentiated pluripotent cells derived from dental pulp cells. Active cells and black triangles indicate oral mucosal cells, respectively.
As shown in FIGS. 1 to 4, DSPP, DMP1, and DSP in dental pulp cells and dental pulp cells were all detected at a high level about two weeks after the start of culture. These showed higher values than those of oral mucosal cells. This suggests that pulp cells and dental pulp cells were undifferentiated cells after the start of culture, but differentiated into odontoblasts by culturing, compared to oral mucosal cells. Means that the cells are distinctly different from epithelial cells.
In particular, it was shown that dental pulp cells were higher in susceptibility to osteoblast-inducing factors than dental pulp cells. This suggests that the dental pulp cells are more undifferentiated than the dental pulp cells and have higher dentin forming ability.
As described above, it was shown that the cell group derived from dental pulp cells and dental pulp cells contains undifferentiated pluripotent cells that can be differentiated into other cell lineages.
[0024]
2. Comparison of undifferentiated pluripotent cells derived from tooth-related cells with myeloid stem cells Then, using dental pulp cells as the tooth-related cells of the present invention, the pluripotency in dental pulp cells was compared with bone marrow-derived mesenchymal stem cells did.
Dental pulp cells were collected as described above. On the other hand, myeloid stem cells were isolated and identified by collecting bone marrow fluid from the iliac crest. Details of this method are well known in the art. The dental pulp tissue is visually considered to be an internal tissue surrounded by the hard tissue of the tooth. Therefore, the dental pulp tissue was visually identified using an extracted tooth or the like and collected.
Alkaline phosphatase (ALP) activity was used as an index of hard tissue formation. ALP activity was calculated as a value per complex. Using 5 μl of the supernatant, the solution was added dropwise to 1 ml of 50 mM p-nitrophenyl phosphate (containing 1 mM MgCl ₂ ). After that, the mixture was reacted at 37 ° C. for 30 minutes, stopped after the reaction with 0.2 N sodium hydroxide, and the absorbance at 410 nm was measured.
FIG. 5 shows the results of myeloid stem cells (□) and those after induction (○), undifferentiated pluripotent cells derived from dental pulp cells (solid triangles), those after induction (●), and oral mucosal cells (×). Shown in
[0025]
As shown in FIG. 5, both the dental pulp cells and the mesenchymal stem cells induced into osteoblasts by the inducer have increased DMP1 activity around 18 days after the start of the culture (see FIG. 5 (A)). ALP activity also increased (see FIG. 5 (B)).
This suggests that pulp cells were induced to differentiate into osteoblasts by bone marrow-derived mesenchymal stem cells, as well as bone marrow-derived mesenchymal stem cells. It is considered to have the property.
Rather, in terms of ALP activity, osteoblasts derived from dental pulp cells show higher activity than osteoblasts derived from mesenchymal stem cells, and undifferentiated pluripotent cells derived from dental pulp cells show bone formation. Was suggested to be more useful than mesenchymal stem cells.
[0026]
3. Preparation of related tissue or tooth by undifferentiated pluripotent cells Further, formation of related tissue or tooth by mixing dental pulp cells with a carrier matrix was examined.
Β-TCP was used as the carrier matrix, and the same osteoblast-inducing factor as described above was used. These were mixed with dental pulp cells at a concentration of 10 ⁶ cells / ml for β-TCP (a cylindrical block having a diameter of 5 mm and a height of 4 mm, a pore diameter of 200 to 400 μm, and a porosity of 90%), and were mixed at 37 ° C., 5% After subculture for 20 days under CO ₂ , the cells were implanted subcutaneously at the back of nude mice.
[0027]
Sixteen days after transplantation, microscopic findings revealed hard tissue-like tissue (dentin-like tissue: astrocytes) in the cavity of the carrier, indicating that dentin was formed. This suggests that undifferentiated pluripotent cells derived from dental pulp cells have stronger tooth forming ability when mixed with β-TCP.
The results suggest that, in addition to regenerating and restoring tissue from a portion of the missing tooth, individual tooth fragments can be made and used for tooth restoration.
[0028]
Therefore, the undifferentiated pluripotent cell of the present invention can be easily obtained from an extracted tooth, which is usually discarded, and can be differentiated into cells of another lineage. It is. In addition, by using such undifferentiated pluripotent cells, a large amount of tooth restoration fragments and the like can be prepared, and can be effectively used for treatment of teeth or tissues around teeth. Furthermore, a tissue related to undifferentiated pluripotent cells can be prepared in a manner similar to that of a tooth, and can be effectively used for various treatments requiring tissue donation.
[0029]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the undifferentiated pluripotent cell which can be obtained noninvasively and easily, and a highly useful transplant using this can be provided.
[0030]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a schematic diagram showing dental pulp cells and dental pulp cells according to the present invention.
FIG. 2 is a graph illustrating DSPP activity of dental pulp cells and the like according to an example of the present invention.
FIG. 3 is a graph illustrating DMP1 activity of dental pulp cells and the like according to an example of the present invention.
FIG. 4 is a graph illustrating DSP activity of dental pulp cells and the like according to an example of the present invention.
FIG. 5 (A) is a graph illustrating DMP1 activity of dental pulp cells and the like according to an example of the present invention, and FIG. 5 (B) is a graph illustrating ALP activity of the same cells as (A).

Claims (6)

Undifferentiated pluripotent cells derived from tooth-related cells. The undifferentiated pluripotent cell according to claim 1, wherein the tooth-related cell is at least one of a dental sac cell and a dental pulp cell. The undifferentiated pluripotent cell according to claim 1 or 2, wherein the undifferentiated pluripotent cell has dentin-forming ability. A method for producing a related tissue or a tooth, comprising obtaining an undifferentiated pluripotent cell from a tooth-related cell and producing a related tissue or a tooth from the obtained undifferentiated pluripotent cell. The method for producing a related tissue or tooth according to claim 4, wherein the tooth-related cell is at least one of a tooth sac cell and a dental pulp cell. The method for producing a related tissue or tooth according to claim 4 or 5, comprising mixing the undifferentiated pluripotent cell, an osteoblast-inducing factor, and a carrier matrix.

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