JP2010268715A - Use of gene expression group characteristic of dental pulp stem cell of milk tooth - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a useful technology in the utilization of dental pulp stem cells of milk and permanent teeth through elucidation of characteristics and differences thereof. <P>SOLUTION: A method for culturing a dental pulp stem cell of milk tooth is provided, comprising a step (step (1)) of providing and culturing a dental pulp stem cell of milk tooth and a step (step (2)) of detecting the expression level of one or more factors selected from the group consisting of FGF2, TGF-beta, collagen I and collagen III regarding the dental pulp stem cell of milk tooth during culturing and of judging the quality of the cell on the basis of the detection results. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a technique useful in utilizing dental pulp stem cells. Specifically, the present invention relates to a culturing technique for deciduous dental pulp stem cells, a quality inspection method for deciduous dental pulp stem cells, and a method for modifying permanent dental pulp stem cells.

  Regenerative medicine using stem cells has attracted attention as a general-purpose alternative technique for diseases difficult to treat with conventional medicine. So far, various stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), somatic stem cells have been found. ES cells and iPS cells have many problems to be solved, such as ethical problems, immune rejection, and canceration. On the other hand, somatic stem cells, especially mesenchymal stem cells (MSCs), have been isolated from various tissues such as bone marrow, adipose tissue, skin, umbilical cord, and placenta (reference documents 1 to 4). It has been reported. Also in the research group of the present inventors, bone marrow-derived mesenchymal stem cells (BMMSCs) have been applied to the treatment of bone defects, and good results have been obtained (reference documents 5 to 7). However, bone marrow collection is highly invasive, and the burden on donors is large in this era toward an aging society. Moreover, bone marrow-derived mesenchymal stem cells have a problem that the number, proliferation ability and differentiation ability decrease with age (Reference Document 8).

  On the other hand, dental pulp is attracting attention as a readily available source of stem cells. As a new stem cell population with both self-proliferating ability and multipotency, dental pulp stem cells (DPSCs) of permanent teeth and stem cells from exfoliated deciduous teeth (SHED) were identified (reference) Literature 9, 10). According to past reports, DPSCs have the ability to differentiate into odontoblasts and osteoblasts and can form dentin / dental pulp complexes (refs. 9-12). In addition, the effectiveness of DPSCs for systemic diseases such as neurological diseases, heart diseases and ischemic diseases has been reported (Non-Patent Documents 13 to 15). In addition, deciduous teeth fall off naturally as a child and are usually discarded as they are. Therefore, the use of deciduous dental pulp stem cells has the great advantage that there is no invasive problem associated with collection and no ethical problems when using them.

  In addition, as prior art documents related to dental pulp stem cells, documents reporting superior stem cells in permanent teeth and children's teeth (deciduous teeth) (patent document 1, non-patent document 1), mixing pulp cells with carrier matrix Then, the literature (patent document 2) which reports that dentin was formed is enumerated below.

International Publication No. 2006/010600 Pamphlet JP 2004-201612 A

Miura M. et al., SHED: Stem cells from human exfoliated deciduous teeth, PNAS, May 13, 2003, vol.100, no.10, 5807-5812

  Dental pulp stem cells have received much attention in recent years. In the future, in order to promote clinical application of dental pulp stem cells, it is desired to clarify the characteristics and establish an effective usage. It is also important to make efficient use. However, it is difficult to say that the characteristics of dental pulp stem cells are well understood. In particular, the difference between deciduous dental pulp stem cells and permanent dental pulp stem cells has not been sufficiently analyzed and there are many unclear points. On the other hand, deciduous dental pulp stem cells are cells present in the deciduous dental pulp that fall out as a child and are subject to greater restrictions in terms of supply compared to permanent dental pulp stem cells. One of the challenges.

  Accordingly, an object of the present invention is to clarify the characteristics and differences between deciduous dental pulp stem cells and permanent dental pulp stem cells, and to provide techniques (culture methods, quality inspection methods, etc.) useful for using these cells.

Under the above problems, the present inventors conducted detailed analysis focusing on proliferation ability and cell surface markers in order to clarify the characteristics of deciduous dental pulp stem cells and permanent dental pulp stem cells. In addition, the gene expression in deciduous dental pulp and permanent dental pulp was comprehensively examined using microarray analysis. Specifically, gene categories in which gene expression fluctuations were significantly detected, pathway analysis, and stem cell marker gene expression were compared. In addition, quantitative analysis of gene expression was performed and further studies were added. As a result, the features and differences between deciduous dental pulp stem cells and permanent dental pulp stem cells were clarified. In addition, pathways supporting the high proliferative ability of deciduous dental pulp stem cells were found, and factors (growth factors, etc.) contained in the pathway were identified. The present invention described below is based on these results.
[1] A method for culturing deciduous dental pulp stem cells comprising the following steps (1) and (2):
(1) A step of preparing and cultivating deciduous dental pulp stem cells;
(2) The expression level of one or more factors selected from the group consisting of FGF2, TGF-β, collagen I and collagen III is detected for the deciduous dental pulp stem cells in culture, and the cell quality is determined based on the detection results. Step to do.
[2] The culture method according to [1], wherein a high expression level detected is an indicator of high quality.
[3] The culture method according to [1] or [2], wherein in step (2), the expression level is detected for all of FGF2, TGF-β, collagen I and collagen III.
[4] The culture method according to any one of [1] to [3], further comprising the following step (3):
(3) A step of examining the presence or absence of expression of one or more cell surface markers selected from the group consisting of CD13, CD29, CD44 and CD73 for the deciduous dental pulp stem cells in culture.
[5] The culture in step (1) is selected from the group consisting of FGF2, TGF-β, collagen I, collagen III, FGF2 inducer, TGF-β inducer, collagen I inducer and collagen III inducer. The culture method according to any one of [1] to [4], which is performed in the presence of the above substance.
[6] One or more selected from the group consisting of FGF2, TGF-β, collagen I, collagen III, FGF2 inducer, TGF-β inducer, collagen I inducer and collagen III inducer, prepared from deciduous dental pulp stem cells Culturing in the presence of the substance of:
[7] A method for determining the quality of deciduous dental pulp stem cells, comprising the following steps (1) and (2):
(1) a step of preparing deciduous dental pulp stem cells;
(2) A step of detecting the expression level of one or more factors selected from the group consisting of FGF2, TGF-β, collagen I and collagen III for the deciduous dental pulp stem cells, and determining the quality of the cells based on the detection results.
[8] The quality determination method according to [7], wherein a high expression level detected is an indicator of high quality.
[9] The quality determination method according to [7] or [8], wherein in step (2), expression levels are detected for all of FGF2, TGF-β, collagen I and collagen III.
[10] The quality determination method according to any one of [7] to [9], further including the following step (3):
(3) A step of examining the presence or absence of expression of one or more cell surface markers selected from the group consisting of CD13, CD29, CD44 and CD73 for the deciduous dental pulp stem cells.
[11] A method for enhancing the proliferation ability of permanent dental pulp stem cells, characterized by genetic modification so that one or more factors selected from the group consisting of FGF2, TGF-β, collagen I and collagen III are highly expressed.
[12] A method for culturing permanent dental pulp stem cells, comprising the following steps (1) and (2):
(1) preparing permanent dental pulp stem cells;
(2) Culture in the presence of one or more substances selected from the group consisting of FGF2, TGF-β, collagen I, collagen III, FGF2 inducer, TGF-β inducer, collagen I inducer and collagen III inducer Step to do.
[13] The culture method according to [12], wherein the permanent dental pulp stem cells are permanent dental pulp stem cells having an increased proliferation ability obtained by applying the method according to [11].
[14] Deciduous dental pulp stem cells cultured by the culture method according to any one of [1] to [6], and the quality was confirmed by the quality judgment method according to any one of [7] to [10] A therapeutic cell comprising a deciduous dental pulp stem cell or a permanent dental pulp stem cell cultured by the culture method described in [12] or [13].
[15] The therapeutic cell according to [14], which is used for regeneration of bone tissue, cartilage tissue, nerve tissue, skin tissue, hair tissue, periodontal tissue or vascular tissue, or for treatment of a systemic disease.
[16] A therapeutic composition comprising the therapeutic cell according to [14] or [15] as a constituent component.

(A) to (C): SHED cell morphology (A), DPSCs cell morphology (B), BMMSCs cell morphology (C). (D) to (F): immunostaining of stem cell marker STRO-1. SHED (D), DPSCs (E), and BMMSCs (F) were all STRO-1 positive (green fluorescence). The nucleus was visualized using DAPI (blue fluorescence in the original image). (G): The growth rates of SHED, DPSCs and BMMSCs were compared using BrdU. The growth rate of SHED is significantly higher than that of DPSCs and BMMSCs. Bars indicate standard deviation (SD). * P <0.05 was considered significant. Result of pathway analysis. In comparison with DPSCs, SHED shows a remarkable pathway. In SHED, the pathway of cell proliferation is activated. The square on each gene represents the strength of expression. The darker the expression, the stronger the expression (in the original image, the expression increases in red and the expression decreases in blue). Real-time PCR results. The expression levels of genes involved in cell proliferation pathways were examined. Compared with DPSCs, SHED showed significantly higher expression of FGF2, TGF-β, Col I and Col III (p <0.05).

As shown in the examples described later, as a result of the study by the present inventors, it was found that deciduous dental pulp stem cells expressed significantly higher FGF2, TGF-β, collagen I and collagen III than permanent dental pulp stem cells. did. Moreover, expression of CD13, CD29, CD44 and CD73, which are mesenchymal stem cell markers, was observed in the deciduous dental pulp stem cells. The first aspect of the present invention provides a method for culturing deciduous dental pulp stem cells based on these findings. The culture method of the present invention includes the following steps (1) and (2).
(1) Step of preparing and cultivating deciduous dental pulp stem cells (2) Expression level of one or more factors selected from the group consisting of FGF2, TGF-β, collagen I and collagen III for the deciduous dental pulp stem cells in culture And detecting cell quality based on the detection result

  In step (1), first, deciduous dental pulp stem cells separated from a living body are prepared. Deciduous dental pulp stem cells are collected from the pulp of the deciduous tooth. An example of collection and subsequent culture operation is shown in the following (i) to (iii). In addition, although the deciduous dental pulp stem cell of the state isolate | separated from the biological body is prepared, the state in which other cells were mixed may be sufficient. Of course, the present invention may be applied to a dental pulp stem cell in which no other cells are mixed or substantially mixed by performing a separation process.

(I) Collection of dental pulp After the fallen deciduous or extracted deciduous teeth are immersed in a chlorohexidine or isodine solution, they are collected in a medium. Subsequently, under raw water injection, the deciduous teeth or extracted deciduous teeth are divided as necessary using a dental bar. The dental pulp is collected using a dental file and collected in the medium.

(Ii) Enzyme treatment and cell seeding Collagenase or dispase is allowed to act on the recovered dental pulp. For example, 3 mg / ml collagenase and 4 mg / ml dispase are added to the medium and left at 37 ° C. for 1 hour. After the enzyme treatment in this way, contaminant components are removed through a cell strainer. After washing the cells, seed them in a culture vessel. The culture vessel is transferred into an incubator and cultured (37 ° C., 5% CO ₂ ). As the culture solution, for example, DMEM (Dulbecco's Modified Eagle's Medium) added with serum or the like can be used. Specific examples of the culture solution are DMEM supplemented with fetal calf serum (20%), penicillin (100 U / ml), streptomycin (100 μg / ml) and amphotericin B (0.25 μg / ml). A culture dish, an Erlenmeyer flask, etc. are used as a culture container. In this case, serum is not necessarily required.

(Iii) Selective culture of adherent cells In this step, adherent cells are first selected. Adherent cells can be selected by removing floating components. Removal of floating cells can be easily performed by exchanging the medium. Specifically, a part or all of the medium is exchanged by sucking and removing part or substantially all of the medium and then pouring a new medium into the culture vessel. This medium exchange operation may be repeated a plurality of times. In order to sufficiently remove floating components, it is preferable to perform medium exchange at 3 to 4 times / week.

  Adherent adherent cells (cells adhered to the culture vessel) remaining by removing floating components are subjected to further culture. The culture here can be performed under the same conditions as in (ii). During culture, the medium is changed as appropriate. For example, the medium is changed once every three days.

Subculture (expansion culture) may be performed after the cells have grown to some extent. For example, when observing with the naked eye, it reaches the sub-confluent state (a state in which the cells occupy about 70% of the surface of the culture container) or confluence, and the cells are detached from the culture container and collected, and again the culture filled with the culture solution Seed in containers. Subculturing may be repeated. For example, the subculture is performed 1 to 3 times to grow to the required number of cells (for example, about 1 × 10 ⁷ cells / ml). The cell can be detached from the culture vessel by a conventional method such as trypsin treatment.

  The culture conditions are not particularly limited as long as the growth and proliferation of deciduous dental pulp stem cells is promoted. As the culture solution, for example, DMEM (Dulbecco's Modified Eagle's Medium) added with serum, growth factors, antibiotics, or the like can be used.

  In a preferred embodiment, in view of the fact that deciduous dental pulp stem cells highly express FGF2, TGF-β, collagen I and collagen III, FGF2, TGF-β, collagen I are promoted to promote cell quality maintenance and efficient proliferation. Culturing is carried out in the presence of one or more substances selected from the group consisting of collagen III, FGF2 inducer, TGF-β inducer, collagen I inducer and collagen III inducer. Preferably two or more, more preferably three or more, and still more preferably four or more substances are used in combination.

  The “inducing substance” is a general term for substances having an action of promoting expression of a target (in the present invention, FGF2, TGF-β, collagen I or collagen III). For example, an FGF2-inducing substance is a substance that promotes the expression of FGF2, and when this substance is added to the medium, the FGF2 expression level of the cells increases. The increase in the expression level can be confirmed by detecting the target mRNA level extracted and purified from the cell lysate or by detecting the target level in the medium.

  Examples of the inducer include a substance located upstream of the target on the pathway or the signal path. A specific example of an FGF2 inducer is FGF1, a specific example of a TGF-β inducer is FGF2, a specific example of a collagen I inducer is CTGF (connective tissue growth factor), and a specific example of a collagen III inducer is CTGF.

  In step (2), the expression level of a specific factor is detected for the deciduous dental pulp stem cell in culture, and the quality of the cell is determined using the result. The “specific factors” here are FGF2, TGF-β, collagen I and collagen III. The expression level of one or more of these factors will be detected, but preferably the expression level is detected for two or more, more preferably three or more, and most preferably all of them. Usually, the greater the number of factors to be detected, the higher the reliability of the determination result.

  “Expression level” is used as a term encompassing gene expression level and protein expression level. Therefore, in the present invention, the expression state of a specific factor is detected at the gene level or protein level. In the former case, the mRNA of the factor to be detected may be detected by, for example, RT-PCR. In the latter case, detection can be performed using an antibody or the like that shows specific binding properties to the detection target factor. As the sample, a part of the culture solution or a part of the cell population is used. In addition, you may decide to measure both a gene level and a protein level, and to perform comprehensive evaluation or evaluation confirmation.

  In the present invention, a high expression level detected is an indicator of high quality. That is, if the expression level is high as a result of detection, it is determined that the cells in culture are of high quality (in other words, high quality is maintained). When such a positive determination is made, for example, (a) the culture is continued or (b) the cells in the culture are preserved. Continuation of culture is effective when cells need to be grown at this stage, while cell storage (storage conditions, eg -198 ° C) is useful when cells need not be grown at this stage or in the future. This is effective when maintaining the quality of cells without degrading as much as possible in preparation for use. Cells obtained from various donors may be stored in the form of dental pulp stem cell banks.

  On the other hand, if the negative result in step (2), that is, if the expression level is found to be low, for example, (a) suspending the culture or (b) performing a process for increasing the expression level. Become.

  In one embodiment of the present invention, the expression level in step (2) is detected at two or more time points with different elapsed times from the start of culture. In this aspect, typically, it is determined whether or not the quality of the cell is maintained based on the comparison with the previous detection result, but the present invention is not limited to this. For example, the comparison with the detection result of the previous time is performed. It may be used to determine the quality. Alternatively, the expression level may be detected over time to monitor changes in cell quality. This aspect is particularly effective when the culture is continued for a long period of time, for example, when the subculture is repeated.

By the way, as shown in the Examples described later, it was shown that the dental pulp stem cells expressed CD13, CD29, CD44 and CD73, which are mesenchymal stem cell surface markers. Based on this knowledge, it can be said that these cell surface markers are useful as an indicator that the quality of the dental pulp stem cells in culture is maintained. Therefore, in one embodiment of the present invention, the following step (3) is additionally performed, and the quality of the cell is determined using the result together with the expression level detected in step (2).
(3) A step of examining the presence or absence of expression of one or more cell surface markers selected from the group consisting of CD13, CD29, CD44 and CD73 for the deciduous dental pulp stem cells in culture

  Cell surface markers CD13, CD29, CD44, and CD73 are mesenchymal stem cell markers that are also expressed in bone marrow mesenchymal stem cells (BMMSCs). Confirming the expression of these cell surface markers is useful in ensuring that the cells in culture maintain the characteristics of stem cells. The presence or absence of expression of one or more of these cell surface markers is examined, but preferably two or more, more preferably three or more, and most preferably all of these are examined for the presence or absence of expression. Usually, the greater the number of cell surface markers to be examined, the higher the reliability of the determination result.

  The presence or absence of expression of the cell surface marker may be examined according to a conventional method. For example, the presence or absence of expression of a specific cell surface marker can be easily examined by using flow cytometry.

The second aspect of the present invention provides a method for determining the quality of deciduous dental pulp stem cells based on the knowledge that deciduous dental pulp stem cells highly express FGF2, TGF-β, collagen I and collagen III. The quality judgment method of the present invention includes the following steps (1) and (2).
(1) Step of preparing deciduous dental pulp stem cells (2) Detecting the expression level of one or more factors selected from the group consisting of FGF2, TGF-β, collagen I and collagen III for the deciduous dental pulp stem cells To determine cell quality based on

For the details of steps (1) and (2) here, the corresponding explanation of the first aspect is incorporated. Also in this quality determination method, a high expression level detected is an indicator of high quality. Further, the greater the number of factors to be detected, the higher the reliability of the determination result. In the most preferred mode, the expression levels of all these factors are detected. On the other hand, it is preferable to further carry out the following step (3) as in the first aspect, and to determine the quality of the cells in consideration of the presence or absence of expression of a specific cell surface marker.
(3) The step of examining the presence or absence of expression of one or more cell surface markers selected from the group consisting of CD13, CD29, CD44 and CD73 for the deciduous dental pulp stem cells

  Here, as shown in the Examples described later, in addition to the fact that deciduous dental pulp stem cells have a higher proliferative ability than permanent dental pulp stem cells, deciduous dental pulp stem cells significantly express FGF2, TGF-β, collagen I and collagen III. It was revealed that these factors have an advantageous effect on the improvement of the growth ability. Therefore, the third aspect of the present invention is a gene modification so that one or more factors selected from the group consisting of FGF2, TGF-β, collagen I and collagen III are highly expressed as a means for effectively utilizing permanent dental pulp stem cells. A method for enhancing the proliferation ability of permanent dental pulp stem cells is provided. When the method of the present invention is applied, a feature related to the high proliferative ability of the deciduous dental pulp stem cell is imparted to the permanent dental pulp stem cell, and as a result, a permanent dental pulp stem cell with improved proliferative ability is obtained. Permanent dental pulp stem cells are relatively easy to obtain, but have a lower proliferation ability than deciduous dental pulp stem cells. The method of the present invention removes the disadvantages and increases the clinical usefulness of permanent dental pulp stem cells, and its significance is very great. In the method of the present invention, genetic modification is performed so that the target (FGF2, TGF-β, collagen I or collagen III) or the target inducer is forcibly expressed. Typically, a vector into which an expression cassette containing an appropriate gene (a gene encoding a target or a target inducer) is inserted may be introduced into permanent dental pulp stem cells. Methods and techniques for gene modification, such as vector construction methods and gene transfer methods, are well known, such as Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) or Current protocols in molecular biology (edited by Frederick M Ausubel et al., 1987) is helpful. The method for collecting and culturing permanent dental pulp stem cells is the same as the method for collecting and cultivating deciduous dental pulp stem cells.

The present invention further provides, as a fourth aspect, a method for culturing permanent dental pulp stem cells comprising the following steps (1) and (2).
(1) Step of preparing permanent dental pulp stem cells (2) FGF2, TGF-β, collagen I, collagen III, FGF2 inducer, TGF-β inducer, collagen I inducer and collagen III inducer are selected from the group Culturing in the presence of one or more substances

  In the culturing method of the present invention, the growth rate of permanent dental pulp stem cells is improved by adding to the medium a factor that has been confirmed to be significantly highly expressed in deciduous dental pulp stem cells exhibiting high proliferation ability. The method for collecting and culturing permanent dental pulp stem cells in the culture method is the same as the method for collecting and cultivating deciduous dental pulp stem cells. In addition, you may decide to use the permanent dental pulp stem cell by which the method of the said 3rd aspect was applied obtained by which the proliferation ability was improved as a cell which uses for the said culture | cultivation method.

  Examples of the use of dental pulp stem cells obtained by applying the culture method of the present invention (first and fourth aspects) or the quality judgment method of the present invention (second aspect) are bone tissue by autotransplantation or allograft , Cartilage tissue, nerve tissue, skin tissue, hair tissue, periodontal tissue or vascular tissue regeneration, and systemic diseases (Parkinson's disease, heart disease, neurological disease, spinal cord disease, diabetes, bone disease, cartilage disease, dental Treatment of diseases, etc.). Therefore, the present invention provides, as a further aspect, a therapeutic cell comprising a dental pulp stem cell cultured by the culture method of the present invention or a dental pulp stem cell whose quality has been confirmed by the quality determination method of the present invention, and a treatment comprising the dental pulp stem cell as a main component A composition is provided. Here, “for treatment” means regeneration of bone tissue, cartilage tissue, nerve tissue, skin tissue, hair tissue, periodontal tissue, vascular tissue, etc., systemic diseases (Parkinson's disease, heart disease, neurological disease, spinal cord disease) , Diabetes, bone disease, cartilage disease, dental disease, etc.), and the target disease or disease state is not particularly limited. The term “dental pulp stem cell” is a term encompassing deciduous dental pulp stem cells and permanent dental pulp stem cells.

In one aspect, the dental pulp stem cells are differentiated and induced into a specific cell lineage and then used as the therapeutic cell of the present invention or as a constituent of the therapeutic composition of the present invention. For example, if a therapeutic cell or therapeutic composition intended to regenerate bone tissue is prepared, an induction treatment is performed so as to differentiate into a bone cell. In a typical procedure, three additives are added to the culture medium: dexamethasone (Dex), sodium β-glycerophosphate (β-GP), and L-ascorbic acid diphosphate (AsAP). (Or by replacing with a medium (osteoinduction medium) containing these additives) to promote differentiation into bone cells. The addition amount of these additives is, for example, about 10 mM for dexamethasone, about 10 ⁻⁸ M for sodium β-glycerophosphate, and about 0.05 mM for L-ascorbic acid diphosphate. Medium exchange is performed appropriately during differentiation induction. For example, the medium is changed once every three days. The culture for inducing differentiation is continued, for example, for 1 day to 14 days.

  In one embodiment, the therapeutic composition of the present invention comprises platelet rich plasma in addition to cellular components. Here, “platelet-rich plasma”, that is, PRP (Platelet-rich Plasma) is plasma rich in platelets. In other words, it refers to plasma in which platelets are concentrated. For example, PRP can be prepared by subjecting collected blood to centrifugation according to the method of Whitman et al. (Dean H. Whitman et al .: J Oral Maxillofac Surg, 55, 1294-1299 (1997)). it can. PRP is known to contain abundant growth factors such as Platelet-derived Growth Factor (PDGF), Transforming growth factor β1 (TGF-β1), Transforming growth factor β2 (TGF-β2) (Jarry J. Peterson: Oral surg Oral Med Oral Pathol Oral Radiol Endod, 85, 638-646 (1998)).

  A further embodiment of the therapeutic composition includes cytokines in addition to cellular components. Preferably, in addition to multi-plasma plate plasma, a cytokine is included. As cytokines, for example, BMP, PDGF, bFGF and the like are used. Two or more kinds of cytokines may be used in combination.

  If PRP and / or cytokines are used, the regeneration effect can be enhanced. PRP is also effective for adjusting fluidity and viscosity. That is, if PRP is used, it is possible to prepare the therapeutic composition of the present invention in a gel suitable for transplantation.

  It does not preclude the additional use of other components provided that the expected effect of the therapeutic composition of the present invention (ie, tissue regeneration occurs at the application site) is retained. The components that can be additionally used in the present invention are listed below.

(A) Inorganic bioabsorbable material and organic bioabsorbable material The type of inorganic bioabsorbable material is not particularly limited, but β-tricalcium phosphate (“β-TCP), α-tricalcium phosphate ( α-TCP), tetracalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate can be used.These materials can be used alone or of course. Two or more selected from the above may be used in combination, preferably either β-TCP or α-TCP, or a combination thereof in any proportion, more preferably β-TCP is inorganic bioabsorbable. Inorganic bioabsorbable materials can be obtained by known methods, and commercially available inorganic bioabsorbable materials can also be used. The β-TCP, for example, can use those made of Olympus Optical Co., Ltd..

  The inorganic bioabsorbable material is preferably in the form of a powder having a particle diameter such that the composition of the present invention becomes fluid when used. The powdered inorganic bioabsorbable material can be prepared by crushing and pulverizing the inorganic bioabsorbable material processed to an appropriate size until a desired particle diameter is obtained. The average particle diameter of the inorganic bioabsorbable material is preferably 0.5 μm to 50 μm. More preferably, an inorganic bioabsorbable material having an average particle size of 0.5 μm to 10 μm is used. Even more preferably, an inorganic bioabsorbable material having an average particle diameter of 1 μm to 5 μm is used. It is also possible to use a combination of a plurality of types of inorganic bioabsorbable materials having different particle diameters. The inorganic bioabsorbable material is preferably contained in an amount of 30% to 75% by weight based on the entire composition of the present invention.

  In addition, the fluidity | liquidity of the composition of this invention can be adjusted with the particle diameter and content rate of an inorganic type bioabsorbable material, and desired fluidity | liquidity can be obtained by adjusting both suitably. Moreover, when adding the below-mentioned thickener, fluidity | liquidity can be adjusted also with the addition amount of a thickener.

  As the organic bioabsorbable material, hyaluronic acid, collagen, fibrinogen (for example, Bolheel (registered trademark)) or the like can be used.

(B) Gelling material For example, thrombin and calcium chloride can be added to constitute the composition of the present invention. By adding these, thrombin acts on fibrinogen in PRP to produce fibrin. And viscosity increases by the aggregation action of fibrin. The type of the gelling agent is not particularly limited, and those that act on the components in PRP to increase the viscosity as described above or those that exhibit a thickening effect by themselves can be appropriately selected and used.

  In addition to the gelling material described above, a second gelling material that acts after application (after transplantation) to change the fluidity (viscosity) of the composition of the present invention can also be used in combination. With such a configuration, transplantation is easy because it has appropriate fluidity at the time of use, and after application, the viscosity increases to improve the fixability at the application site, and the mesenchymal tissue (bone) , Cartilage, ligaments, muscles, nerves, teeth or periodontal tissue) can be effectively repaired or regenerated. Moreover, it is not necessary to mold into the shape of the application site in advance, and versatility is high.

  As the gelling material, a material having high biocompatibility is preferably used. In addition to the above examples, hyaluronic acid, collagen, fibrin glue, or the like can be used. Various types of hyaluronic acid and collagen can be selected and used, but it is preferable to employ one suitable for the application purpose (application tissue) of the composition of the present invention. For the purpose of regenerating bone tissue, for example, type I collagen can be used. The collagen used is preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.).

(C) Thickener The fluidity of the composition of the present invention can also be adjusted by adding a thickener. As the thickener, thickening polysaccharides such as sodium alginate, glycerin, petrolatum and the like can be used, but from the viewpoint of safety and / or bone forming ability, the biocompatibility is high and the bioabsorbable or biocompatible It is preferable to use a decomposable one. By adding glycerin or the like, the effect of preventing frost damage can be obtained.

(D) Solvent The composition of the present invention may contain an aqueous solvent. As the aqueous solvent, sterilized water, physiological saline, a buffer solution such as a phosphate solution, or the like can be used. The prepared cells can be suspended in physiological saline or PBS (phosphate buffered physiological saline) to obtain the composition of the present invention (not containing other components such as PRP) and applied to the affected area.

(E) Others The composition of the present invention may contain a stabilizer, a preservative, a pH adjuster and the like in addition to the above components. Growth factors, particularly osteoinductive factors (BMP) can also be included.

  Here, an example of the preparation method of the composition of this invention is shown. In the following preparation methods, (i) a step of preparing a thrombin solution, (ii) a step of preparing platelet-rich plasma (PRP), and (iii) a step of mixing and gelling each component are carried out. Hereinafter, each step will be described.

(I) Step of preparing a thrombin solution In this step, a solution containing a predetermined amount of thrombin is prepared. The thrombin concentration in the thrombin solution is not particularly limited, but is set to a concentration at which appropriate gelation can be achieved in step (iii) described later. For example, a thrombin solution containing a thrombin concentration of 100 U / ml to 10,000 U / ml is used. Preferably, the thrombin concentration is about 1000 U / ml. It is preferable to use human thrombin from the viewpoint of safety and immune rejection. For example, thrombin-Yoshitomi (registered trademark) can be used as human thrombin. Alternatively, human thrombin prepared from autologous blood may be used. By causing thrombin to act in the presence of calcium ions, fibrin is produced from fibrinogen in platelet-rich plasma (PRP) and coagulated (gelled). Therefore, if a thrombin solution containing calcium ions is prepared, it is not necessary to add calcium ions separately when mixing the thrombin solution and PRP (step (iii)). For example, it is preferable to prepare a thrombin solution as a 5% to 25% calcium chloride solution. More preferably, a thrombin solution prepared as an approximately 10% calcium chloride solution is used.

(Ii) Step of preparing platelet-rich plasma (PRP) In this step, platelet-rich plasma (PRP) is prepared from blood separated from a living body. PRP can be prepared according to the Japanese Red PC (concentrated platelet) collection method. Specific examples of the method for preparing PRP are shown below. First, an anticoagulant such as sodium citrate is added to the collected blood and left at room temperature for a predetermined time. Thereafter, the mixture is centrifuged under conditions that separate blood cells and buffy coat (for example, about 1,100 rpm for about 10 minutes). This separates into two layers. After collecting the upper layer, it is further centrifuged at about 2,500 rpm for about 10 minutes. The resulting fraction (Platelet-rich Plasma: PRP) is collected. The preparation method of PRP is not limited to the said method, It can prepare by the method which added correction as needed.

  From the viewpoint of toxicity or immune rejection, it is preferable to prepare PRP using the recipient's own blood (that is, the subject to which the therapeutic composition of the present invention is applied). However, PRP is prepared from blood of the same species. May be.

  Although there is no general definition for the number of platelets contained in PRP (platelet concentration rate), plasma containing about 150 to about 1500 times more platelets than the collected blood is defined as PRP in the present invention. Can do.

In the present invention, the “platelet concentration rate” of PRP is represented by the following formula.
Platelet concentration rate (%) = (average platelet count in PRP / average platelet count in whole blood as starting material) x 100

  Therefore, for example, when the average platelet count in PRP is 1,000,000 and the average platelet count in whole blood is 300,000, the platelet concentration rate is about 3333%. Previous studies have shown that the platelet enrichment rate of PRP is related to tissue regeneration effects. Therefore, in order to obtain a higher regenerative effect, the platelet concentration rate ranges from about 150% to about 1500% (usually equivalent to about 240,000 / μL to about 6,150,000 / μL when converted to the average platelet count). It is preferable to use the PRP. More preferably, PRP having a platelet concentration rate in the range of about 300% to about 700% (usually corresponding to about 480,000 / μL to about 2,870,000 / μL when converted to the average platelet count) is used.

  A PRP having a desired platelet concentration can be obtained by appropriately adjusting the conditions of the centrifugal treatment in preparing the PRP. For example, the above-described two-stage centrifugation is performed, and the first centrifugation is performed under conditions of about 500 rpm to about 1500 rpm (for example, 1,100 rpm) for about 5 minutes to about 15 minutes (for example, about 5 minutes). By performing the second stage centrifugation under the conditions of about 2000 rpm to about 5000 rpm (eg, about 2,500 rpm) for about 5 minutes to about 15 minutes (eg, about 5 minutes), the platelet concentration rate is about 300% to about A PRP in the range of 700% can be obtained. Although it is expected that the platelet concentration rate of the PRP finally obtained will vary even if it is processed under the same conditions, depending on the starting material blood and the equipment used, those skilled in the art usually change the above conditions. The conditions for preparing PRP having a desired platelet concentration rate can be found by taking into account the correction of the conditions based on the platelet concentration rate of the obtained PRP. The platelet concentration rate of PRP can be measured using a conventional method (for example, using commercially available Sysmex XE-2100 (Sysmex, Tokyo, Japan)).

  The platelet enrichment rate of PRP used to construct the therapeutic composition of the present invention is as described above. On the other hand, the platelet concentration of the final composition (the therapeutic composition of the present invention) varies depending on the platelet concentration rate of PRP, the use ratio of PRP and other components combined therewith, etc., for example, about 240,000 / μL to About 6,150,000 / μL, preferably about 480,000 / μL to about 2,870,000 / μL. By containing platelets at such a concentration, a good tissue regeneration effect can be obtained. For example, if PRP with a platelet concentration rate in the range of about 300% to about 700% is used, the platelet concentration of the final composition should be adjusted to a range of about 480,000 / μL to about 2,870,000 / μL. Is possible.

(Iii) Step of mixing and gelling each component In this step, the thrombin solution prepared in step (i), the platelet-rich plasma prepared in step (ii), and the dental pulp stem cells prepared by the above preparation method Mix in the presence of calcium ions to gel. At this time, cytokines (BMP, PDGF, bFGF, etc.) can also be mixed together.

  Preferably, air is mixed at a predetermined ratio when these components are mixed. By mixing air, the gelation state can be adjusted (adjustment of fluidity). In addition, in a composition obtained by mixing air, when an appropriate amount of air is present around it when it is transplanted into a living body, an environment suitable for the survival and proliferation of cells in the composition is created. Therefore, a good tissue regeneration effect can be expected.

In the present invention, each component is mixed and gelled, for example, at the following mixing ratio (volume basis).
(a) Thrombin solution: total amount of platelet-rich plasma and dental pulp stem cells: air = 1: 3-7: 0.1-5.0
By mixing at the mixing ratio, a gel-like composition is obtained that has appropriate fluidity from the viewpoint of operability and fixability after transplantation, and that exhibits a good regenerative effect (therapeutic effect).

  Here, the fluidity of the obtained gel-like composition changes according to the mixing ratio of the total amount of platelet-rich plasma and dental pulp stem cells and / or the mixing ratio of air. That is, the fluidity of the resulting gel composition can be adjusted by manipulating the mixing ratio of (a). Specifically, if a composition having a relatively low fluidity is required, the mixing ratio of the total amount of platelet-rich plasma and dental pulp stem cells is small (and / or the mixing ratio of air is small within the above mixing ratio range. ) If a mixing ratio is used and a composition with relatively high fluidity is required, the mixing ratio of the total amount of platelet-rich plasma and dental pulp stem cells is large (and / or the mixing ratio of air within the above mixing ratio range) The mixing ratio may be adopted. In addition, since thrombin solution, platelet-rich plasma, and dental pulp stem cells are components derived from living bodies, there are some fluctuations in properties due to differences in collection sources, etc., which may affect the gelation state. According to the present inventors' previous experience, a composition in a gelled state having the above excellent properties is usually obtained by mixing the respective components within the range of the mixing ratio.

In a preferred embodiment of the present invention, the mixing ratio of each component is as follows.
(a1) Thrombin solution: total amount of platelet-rich plasma and dental pulp stem cells: air = 1: 4-6: 0.3-3.0

By employing the mixing ratio, it is possible to more reliably prepare a gelled composition having a desired fluidity. Specific examples of the mixing ratio of each component are shown below.
Thrombin solution: total platelet-rich plasma and dental pulp stem cells: air = 1: 4: 1.0
Thrombin solution: Total amount of platelet-rich plasma and dental pulp stem cells: Air = 1: 5: 1.0
Thrombin solution: total platelet-rich plasma and dental pulp stem cells: air = 1: 6: 1.0

By mixing each component in the above ratio, a composition containing about 1.0 × 10 ⁵ to about 1.0 × 10 ⁸ / ml of dental pulp stem cells can be typically obtained. According to such a composition, when applied to a tissue defect part, a good tissue regeneration effect can be expected.

  As a method for applying the therapeutic cell or therapeutic composition of the present invention, injection, embedding, filling, or coating can be employed in a tissue defect. If it is prepared in a gel form having an appropriate fluidity, it can be applied by a simple technique such as filling, pouring or coating. Moreover, if it is a gel, it can be easily inserted into the application site using an injection needle or the like (it can also be applied without opening the wound), and is pre-molded according to the shape of the tissue defect. The versatility is high.

  Hereinafter, the present invention will be described in more detail with reference to examples.

1. Materials and Methods (1) Collection and culture of cells Human pulp tissues were collected from the deciduous teeth and permanent teeth of healthy individuals with the approval of the Nagoya University Ethics Committee. The separation method and culture method of SHED and DPSCs were in accordance with the previously reported methods (reference documents 9, 10). Briefly, the pulp was carefully collected and treated in a solution containing 3 mg / ml collagenase I and 4 mg / ml dispase at 37 ° C. for 1 hour. After filtering using a 70 mm cell strainer (Falcon, BD Labware, Franklin Lakes, NJ), 20% mesenchymal cell growth supplement (Lonza® Inc. Walkersville, MD) and antibiotics (penicillin 100 U / ml) The cells were cultured using DMEM (Dulbecco's Modified Eagle's Medium) containing 100 μg / ml streptomycin and 0.25 μg / ml amphotericin B (GIBCO) (37 ° C., 5% CO ₂ ). Human BMMSCs were purchased from Lonza (registered trademark) Inc. and cultured according to the instruction manual.

(2) Analysis of cell growth rate
Using a BrdU staining kit (Invitrogen, Carlsbad, Calif.), The growth rate of SHED, DPSCs, and BMMSCs was evaluated using BrdU (bromodeoxyuridine) uptake (12 hours) as an index (3 samples per test group). For the statistical processing, Tukey-Kramer test based on one-way analysis of variance (ANOVA) was used. A p value <0.05 was considered statistically significant.

(3) Cell surface antigen analysis by FACS The trypsin-treated cultured cells were centrifuged and washed with PBS. Cells were incubated for 45 minutes at 4 ° C. in the presence of various specific antigens. Anti-human CD14 mouse antibody and anti-human CD31 mouse antibody (BD Pharmingen, San Diego, CA) conjugated with FITC (fluorescein isothiocyanate), anti-human CD73 mouse antibody (BD Pharmingen) conjugated with PE (phycoerythrin), APC (Allophycocyanin) -conjugated anti-human CD13 mouse antibody, anti-human CD29 mouse antibody and anti-human CD34 mouse antibody (BD Pharmingen), PerCP-conjugated anti-human CD45 mouse antibody (BD Pharmingen), biotin-conjugated anti-human CD44 Mouse antibody (BD Pharmingen) was used. As a secondary antibody for detecting an anti-human CD44 mouse antibody bound with biotin, Streptavidin (BD Pharmingen) bound with PerCP was used. Evaluation was by flow cytometry using FACS Calivur (BD Pharmingen).

(4) Immunofluorescence staining of STRO-1
SHED, DPSCs and BMMSCs were subcultured in 24-well plates. The cells were fixed by treatment with PBS containing 3% paraformaldehyde for 30 minutes. Subsequently, the cells were washed twice with PBS and then treated with PBS containing 100 mM glycine for 20 minutes. Next, after permeabilization (30 minutes) with PBS containing 0.2% of Triton-X, it was incubated for 20 minutes in PBS containing 5% donkey serum and 0.5% bovine serum albumin. Cells were then incubated with anti-human STRO-1 mouse antibody (primary antibody, 1: 100) (1 hour). Subsequently, the secondary antibody (1: 500) was reacted for 30 minutes and then embedded with DAPI-containing Vectashield (Vector Laboratories Inc., Burlingame, Calif.).

(5) cDNA microarray analysis Total RNA was prepared from SHED and DPSCs derived from the same donor using RNeasy (registered trademark) Mini Kit (QIAGEN, Valencia, CA). Microarray experiments were performed using Agilent Expression Array Whole Human Genome oligo DNA microarray (Agilent, Santa Clara, CA) (consigned to Takara Bio Inc.). This microarray contains 41,078 probe (gene) sets. Experiments were performed using Quick Amp Labeling Kit two color, Gene Expression Hybridization Kit and Gene Expression Wash Pack (Agilent) according to Quick Amp Labeling Kit two color manual (Agilent). Data correction and analysis were performed using Agilent Feature Extraction ver. 9.5 software. Liner / LOWESS (Locally Weighted Liner Regression) was used for data correction. The corrected data was taken into GeneSpring GX 10.0 software (Agilent) for further analysis.

(6) Quantitative real-time RT-PCR
Real-time RT-PCR was performed according to a previously reported method (Reference Document 16). Table 1 shows primers and probes for human fibroblast growth factor 2 (FGF2), transforming growth factor β2 (TGF-β2), type I collagen (Col I) and type III collagen (Col III).

  Primers and probes for GAPDH (TaqMan GAPDH detection reagent) were purchased from PerkinElmer and Applied Biosystems. The relative expression level was corrected by the expression level of GAPDH. Statistical processing was performed by Student's t test, and significant difference was found when p value <0.05.

尚、間葉系細胞マーカー、内皮細胞マーカー、造血系細胞マーカーについてフローサイトメトリーで解析した。平均±標準偏差で陽性率（％）を示した。ａ：n=4、ｂ：n=4、ｃ：n=3。 2. Results (1) Comparison of SHED, DPSCs and BMMSCs characteristics
SHED and DPSCs showed fibroblast-like morphology similar to BMMSCs (FIGS. 1A-C). As a result of immunofluorescence staining, it was found that all of SHED, DPSCs, and BMMSCs contain STRO-1 positive cells (FIGS. 1D-F). Table 2 shows the positive rates of mesenchymal cell markers (CD13, CD29, CD44 and CD73), endothelial cell marker (CD31), hematopoietic cell markers (CD34, CD45) and monocyte marker (CD14).

In addition, mesenchymal cell markers, endothelial cell markers, and hematopoietic cell markers were analyzed by flow cytometry. The positive rate (%) was shown by the average +/- standard deviation. a: n = 4, b: n = 4, c: n = 3.

  Thus, both SHED and DPSCs showed a positive rate of 90% or more for all mesenchymal cell markers. This result shows that SHED has the phenotype of MSCs as well as DPSCs and BMMSCs. SHED showed a higher growth rate than DPSCs and BMMSCs (p <0.05) (FIG. 1G).

(2) Gene expression profiles of SHED and DPSCs Since they show a high growth rate, SHED and DPSCs are considered to be useful for cell-based therapy as an alternative to BMMSCs. Therefore, we focused on SHED and DPSCs and decided to add further studies. A cDNA microarray was used to compare gene expression profiles of SHED and DPSCs. As a result of the analysis, among 41,078 genes, 4,386 genes showed a difference of more than double the expression level between SHED and DPSCs (2,159 genes were increased by SHED and 2,227 genes were decreased by SHED. ).

(3) Gene ontology analysis
GeneSpring GX 10.0 software (Table 3) gene ontology (GO) analysis was performed to determine the category of enriched genes in SHED compared to DPSCs.

  The most prominent GO category was related to extracellular matrix. Genes related to developmental processes, such as genes related to anatomical structures, multicellular organisms, and blood vessels, were also a category of concentrated genes characteristic of SHED.

(4) Pathway analysis Genes to be analyzed were extracted with an expression increase (up-regulation) and expression decrease (down-regulation) with a 2-fold change as a cut-off and a p-value of 0.05 as a cut-off. One of the most significant pathways was cell proliferation. A network of cell proliferation is shown in FIG. Extracellular matrix genes such as collagen (Col I, III, VII, XIII) and proteoglycans (glypican, versican) are included in the cell proliferation pathway. Also, growth of fibroblast growth factor (FGF), transforming growth factor β (TGF-β), connective tissue growth factor (CTGF), nerve growth factor (NGF), etc. and bone morphogenetic protein (BMP) Factors are related to this pathway.

(5) Real-time PCR analysis
In order to confirm the importance of cell proliferation pathway in SHED, the expression pattern of related genes was evaluated using real-time PCR (FIG. 3). Compared with DPSCs, SHED showed significantly higher expression of FGF2, TGF-β, Col I and Col III (p <0.05). This result agrees with the result of microarray analysis.

3. Discussion In this study, the characteristics of SHED were compared with those of DPSCs. It was also compared with BMMSCs that have been regarded as a standard for stem cells used in tissue engineering and regenerative medicine. As a result, it was shown that SHED has a high proliferation ability and is rich in extracellular matrix, and was found to be extremely useful as a cell source for treatment using stem cells. SHED has the ability to differentiate into various cells such as osteoblasts, chondroblasts, adipocytes, and nerve cells (reference documents 10 to 12). Previous reports show that SHED has the ability to form fibrous bone and dentin structures (Refs. 12, 17, 18). On the other hand, DPSCs isolated from permanent dental pulp (Reference 9) are considered to be useful not only for the regeneration of dental tissues but also for the treatment of systemic diseases (References 13 to 15). There are several reports comparing the characteristics of DPSCs and BMMSCs (Refs. 16, 19-21). In the cDNA microarray analysis, DPSCs and BMMSCS show similar gene expression profiles (References 16 and 19), but their pluripotency is different (References 20 and 21). This fact suggests that the characteristics of stem cells are not uniform and depend on their origin (collection source). In previous reports, the difference between SHED and adult DPSCs was that SHED induced bone formation when implanted subcutaneously in immunodeficient mice, whereas DPSCs formed dentin / dental pulp-like structures. (References 9, 10, 18). However, the difference between SHED and DPSC was largely unknown.

  This study revealed that all of SHED, DPSCs, and BMMSCs were positive for STRO-1 (References 10 and 22), which is one of the mesenchymal stem cell markers. The results of FACS analysis showed that both SHED and DPSCs were positive for most mesenchymal stem cell markers, but negative for endothelial cell markers, hematopoietic cell markers, and monocyte markers. The expression pattern was similar to BMMSCs. These results suggest that, like DPSCs, SHED has a mesenchymal stem cell phenotype but no endothelial or hematopoietic cell phenotype.

  Having a high proliferative ability is one of the most important features in somatic stem cells (Reference 23). As a result of experiments using BrdU, SHED showed the highest growth rate among SHED, DPSCs and BMMSC. Moreover, as a result of pathway analysis, the cell proliferation network is activated in SHED as compared with DPSCs, and this is considered to be the reason why SHED exhibits a higher proliferation rate than DPSCs. The results of microarray analysis revealed that SHED highly expresses growth factors such as FGF, TGF-β, CTGF, NGF and BMP associated with this pathway. FGF2 is a cytokine that promotes the growth of various cells such as fibroblasts, vascular endothelial cells, neuroectodermal cells, and osteoblasts. FGF2 also regulates extracellular matrix production in the process of tissue neogenesis and wound healing. It has been reported that FGF2 exhibits a cell proliferation action and a chondroblast differentiation inducing action on DPSCs (Reference Documents 24 and 25). Therefore, FGF2 may promote cell proliferation and extracellular matrix expression even in SHED. The TGF-β family (TGF-β1, TGF-β2, TGF-β3) plays an important role in the regulation of cell proliferation, extracellular matrix formation, differentiation, migration and apoptosis. The presence of all isoforms in dental pulp cells has been observed (Ref. 26). In this experiment, high expression of TGD-β2 and TGF-β3 was observed in SHED compared to DPSCs. TGF-β2 has been reported to stimulate cell proliferation and collagen synthesis (Refs. 27, 28). Considering this, it is expected that TGF-β2 is involved in the regulation of SHED biological activity. CTGF, which is highly expressed in SHED, is a matrix signal molecule involved in TGF-β-induced cell proliferation, matrix synthesis, angiogenesis, migration, and differentiation of MSCs into the osteoblast lineage (Reference 29 ). The results obtained here suggest that TGF-β and CTGF play an important role in the regulation of SHED biological functions.

  As a result of GO analysis, the gene category in which expression was most markedly increased in SHED was related to extracellular matrix (especially collagen). Collagen is a major component of the extracellular matrix and plays an important role in maintaining the biological and structural integrity of the extracellular matrix (Ref. 30). Collagen is also important as a cell scaffold. Therefore, the enhanced production of collagen indicates that SHED is advantageous as a stem cell source in tissue engineering. In addition, as a result of pathway analysis, collagen (Col I, III, VII, XIII) and proteoglycan (glypican, versican) were shown to be involved in the cell proliferation network. This suggests that these extracellular matrix factors promote cell proliferation in SHED.

  The gene expression profile revealed by this study is extremely important for understanding the characteristics of SHED as stem cells, especially high proliferation ability. One of the reasons why MSCs are used in various medical applications such as tissue engineering and cell-based treatment is their high proliferation ability. In view of this, it is a great advantage in clinical application that SHED exhibits high proliferation ability. Furthermore, being rich in extracellular matrix and growth factors is preferable as a stem cell source used for regenerative medicine. The pathways and genes characteristic of SHED revealed this time can be said to support that SHED is a very useful stem cell source clinically.

  According to the present invention, efficient use and maintenance of quality of dental pulp stem cells can be achieved. The present invention is very useful as a technology that supports clinical application of dental pulp stem cells.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

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SEQ ID NO: 1: description of artificial sequence: primer SEQ ID NO: 2: description of artificial sequence: primer SEQ ID NO: 3: description of artificial sequence: probe SEQ ID NO: 4: description of artificial sequence: primer SEQ ID NO: 5: description of artificial sequence: primer SEQ ID NO: 6: description of artificial sequence: probe sequence number 7: description of artificial sequence: primer sequence number 8: description of artificial sequence: primer sequence number 9: description of artificial sequence: probe sequence number 10: description of artificial sequence: primer SEQ ID NO: 11: description of artificial sequence: primer SEQ ID NO: 12: description of artificial sequence: probe

Claims (16)

A method for culturing deciduous dental pulp stem cells comprising the following steps (1) and (2):
(1) A step of preparing and cultivating deciduous dental pulp stem cells;
(2) The expression level of one or more factors selected from the group consisting of FGF2, TGF-β, collagen I and collagen III is detected for the deciduous dental pulp stem cells in culture, and the cell quality is determined based on the detection results. Step to do.   The culture method according to claim 1, wherein a high expression level detected is an indicator of high quality.   The culture method according to claim 1 or 2, wherein in step (2), the expression level is detected for all of FGF2, TGF-β, collagen I and collagen III. The culture method according to any one of claims 1 to 3, further comprising the following step (3):
(3) A step of examining the presence or absence of expression of one or more cell surface markers selected from the group consisting of CD13, CD29, CD44 and CD73 for the deciduous dental pulp stem cells in culture.   One or more substances selected from the group consisting of FGF2, TGF-β, collagen I, collagen III, FGF2 inducer, TGF-β inducer, collagen I inducer and collagen III inducer in step (1) The culture method according to any one of claims 1 to 4, which is carried out in the presence of.   Prepare deciduous dental pulp stem cells, and one or more substances selected from the group consisting of FGF2, TGF-β, collagen I, collagen III, FGF2 inducer, TGF-β inducer, collagen I inducer and collagen III inducer. Culturing in the presence of deciduous dental pulp stem cells. A method for determining the quality of deciduous dental pulp stem cells, comprising the following steps (1) and (2):
(1) a step of preparing deciduous dental pulp stem cells;
(2) A step of detecting the expression level of one or more factors selected from the group consisting of FGF2, TGF-β, collagen I and collagen III for the deciduous dental pulp stem cells, and determining the quality of the cells based on the detection results.   The quality determination method according to claim 7, wherein a high expression level detected is an indicator of high quality.   The quality determination method according to claim 7 or 8, wherein in step (2), expression levels are detected for all of FGF2, TGF-β, collagen I and collagen III. The quality judgment method according to any one of claims 7 to 9, further comprising the following step (3):
(3) A step of examining the presence or absence of expression of one or more cell surface markers selected from the group consisting of CD13, CD29, CD44 and CD73 for the deciduous dental pulp stem cells.   A method for enhancing the proliferative ability of permanent dental pulp stem cells, characterized by genetic modification so that one or more factors selected from the group consisting of FGF2, TGF-β, collagen I and collagen III are highly expressed. A method for culturing permanent dental pulp stem cells comprising the following steps (1) and (2):
(1) preparing permanent dental pulp stem cells;
(2) Culture in the presence of one or more substances selected from the group consisting of FGF2, TGF-β, collagen I, collagen III, FGF2 inducer, TGF-β inducer, collagen I inducer and collagen III inducer Step to do.   The culture method according to claim 12, wherein the permanent dental pulp stem cells are permanent dental pulp stem cells having an increased proliferation ability obtained by applying the method according to claim 11.   The deciduous dental pulp stem cell cultured by the culture method according to any one of claims 1 to 6, the deciduous dental pulp stem cell whose quality has been confirmed by the quality judgment method according to any one of claims 7 to 10, or A therapeutic cell comprising a permanent dental pulp stem cell cultured by the culture method according to 12 or 13.   The therapeutic cell according to claim 14, which is used for regeneration of bone tissue, cartilage tissue, nerve tissue, skin tissue, hair tissue, periodontal tissue or vascular tissue, or treatment of a systemic disease.   The therapeutic composition which uses the therapeutic cell of Claim 14 or 15 as a component.

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