Medium-based method for realizing differentiation from dental tissue stem cells into neurons

Abstract

The present invention relates to the use of a culture medium-based method for inducing specific differentiation of dental tissue stem cells into dopaminergic neurons. The aim of the present invention is to develop cell applications for use in the treatment of neurodegenerative diseases and pharmaceuticals related to said diseases.

Description

Technical Field

[0001] Technical Field to which the Invention Belongs The present invention relates to the use of a medium-based method for specifically inducing the differentiation of dental stem cells into dopaminergic neurons.

Background Art

[0002] Background of the Invention One source of mesenchymal stem cells in the human body is dental tissue. Stem cells isolated from various parts of teeth have been characterized. Examples of these include cells isolated from dental pulp, periodontal connective tissue, and immature impacted teeth [1]. Dental Derived Stem Cells (DDSCs) are attracting attention because they are derived from neuroectodermal cells (neural crest cells) formed from the neural tube of the ectoderm during embryogenesis [2]. These cells have the ability to differentiate into many diverse types of cells and tissues. It has also been found that these cells have the ability to form bone, fat, cartilage, and nerves in addition to tooth formation [1]. Since DDSCs are derived from neural crest cells and show some neuron-specific expression profiles, they are considered a promising cell type for the treatment of neurodegenerative diseases. The incidence of neurodegenerative diseases, in which the loss of function or cell death of neurons increases and occurs irreversibly, is increasing every day. The limited formation of new neurons in the central nervous system, the presence of factors that delay recovery, and the difficulty of surgery due to the structure of the skull are the reasons that make the treatment of this type of disease difficult.

[0003] One of the beneficial methods for treating these diseases caused by nerve cell damage or loss is the application of stem cells [3]. After being applied to the target site by appropriate techniques, stem cells are observed to move to the damaged site, have the ability to differentiate, and stimulate endogenous stem cells to contribute to the damage repair process [4]. To date, tissues such as teeth, bone marrow, blood, cartilage, and fat have been used as sources of adult stem cells [5]. If these stem cells have the ability to differentiate into the cell types lost in neurodegenerative diseases, it becomes possible to create an individual-specific disease model in a laboratory environment using cells prepared from tissues that are easier to obtain from patients than central nervous system tissues [6]. By studying the neurogenic differentiation ability of these diverse stem cells, it becomes possible to determine the cell types and methods that can most contribute to both the replacement of dead nerve cells in the treatment of neurodegenerative diseases and the development of various therapeutic methods through the creation of individual-specific cell culture models of these diseases.

[0004] Differentiation from induced pluripotent stem cells into nerve cells has disadvantages such as low differentiation yield by culture, high mutation rate, higher cost than culturing and differentiating mesenchymal cells, selecting cells containing viral genes, and a relatively high carcinogenicity in in vivo cell transplantation. In addition, since dental stem cells are developmentally close to nerve tissue, it is known that mesenchymal stem cells derived from fat and bone marrow used for neural differentiation take time to differentiate into nerve cells and do not have the potential as high as dental tissue stem cells. Also, mesenchymal stem cells have the disadvantage that it is methodologically difficult to obtain cells, especially from bone marrow and cartilage, and a large amount of cells cannot be obtained. Finally, in the differentiation of stem cells from various sources, when substances such as DMSO, BHA, or β-mercaptoethanol, which damage the morphological structure of cells and cause pseudo-neurogenesis in cells, are used in the culture medium, it becomes impossible to continue culturing the differentiated cells.

[0005] Chinese Patent CN104726406 is a document known in the art, which discloses a method for inducing the differentiation of dental pulp mesenchymal stem cells into nerve cells. In this patent application, when differentiating stem cells into nerve cells, in order to arrest the cell cycle when the cell density is 40-50%, as described in the section of the method of the present invention and shown in Figure 1, chemical substances (VPA and IBMX) are further added. In this way, the cells are differentiated in a healthy state and are induced into the nervous system by the method of the present invention rather than due to high density.

[0006] Chinese Patent CN1590537 is a document known in the art, which discloses a method for isolating and culturing outer mesenteric stem cells. Within the scope of the same invention, this method can be used for bone tissue engineering, muscle tissue engineering, dental tissue engineering, and the repair of peripheral nerve cells. Further, in this invention, in order to reliably regenerate peripheral nerve cells, a differentiation means into the glial system rather than the nervous system is used. Also, the present invention is methodologically different in that only the forskolin chemical substance is used and it is composed of a single step. As is known from this document, it has been shown that it is not sufficient to use a cyclic-amp activator such as forskolin alone in differentiation. At the same time, the reliability of the differentiation method of the present invention has been morphologically proven by cresyl violet staining (Figures 2-3). For the above reasons, it is confirmed that the method of the present invention is more beneficial.

[0007] One of the inventions in the relevant technical field, U.S. Patent Application No. US2016296669, discloses a method for manufacturing a graft material for treating nerve damage. The method of the invention includes the step of culturing dental tissue stem cells in a medium that does not contain growth factors other than FGF2 (or bFGF (Basic Fibroblast Growth Factor)). Furthermore, the FGF2 factor used in the patent document is known to activate only genes related to the nervous system of dental pulp-derived stem cells. However, it is known that many signal transduction pathways, including the cell cycle, play important roles in stem cell differentiation. Therefore, it is clear that even if only the FGF2 factor is administered to stem cells, effective differentiation into nerve cells cannot be obtained. Considering these, the invention according to this patent application, as also described in its method, follows a protocol consisting of two steps to perform both activation of the differentiation pathway and maturation of nerve cells, and as a result, it has been proven to show gene expression of mature functional nerve cells as shown in Figure 4. Summary of the Invention

[0008] Summary of the Invention An object of the present invention is to differentiate dental derived stem cells into dopaminergic neurons by the medium-based method of the present invention.

[0009] Another object of the present invention is to develop the treatment of neurodegenerative diseases and the application of cells for use in drugs related to the diseases.

[0010] Detailed Description of the Invention A "medium-based method for the differentiation of dental stem cells into nerve cells" developed to achieve the object of the present invention is shown in the accompanying drawings. Further aspects of the present invention are described below: [Item 1] A medium-based method for implementing the differentiation of dental tissue stem cells into nerve cells, capable of differentiating mesenchymal stem cells obtained from dental tissue into dopaminergic neurons, comprising - A step of seeding the dental tissue stem cells at a concentration of 5000 cells / cm 2 ; - After culturing for 24 hours, adding the cells to the first neurogenesis induction medium and continuing the medium application for 4 days; - Then, adding the cells to the second neurogenesis induction medium and continuing the medium application for 2 days; - A step of terminating differentiation after 6 days A method characterized by including the above steps. [Item 2] The first neurogenesis induction medium contains - Dmem / F12 with Glutamax added, - 1% B-27 supplement, - 100 μM 3-isobutyl-1-methylxanthine (IBMX), - 2 mM sodium valproate (VPA), - 0.1 μM forskolin, - 20 ng / ml basic fibroblast growth factor (bFGF), - 20 ng / ml epidermal growth factor (EGF) A medium-based method for realizing the differentiation of dental tissue stem cells into nerve cells according to Item 1. [Item 3] The second neurogenesis induction medium contains - Dmem / F12 with Glutamax added, - 1% B-27 supplement - 100 μM 3-isobutyl-1-methylxanthine (IBMX) - 2 mM sodium valproate (VPA) - 0.1 μM forskolin - 20 ng / ml basic fibroblast growth factor (bFGF) - 20 ng / ml epidermal growth factor (EGF) - 30 ng / ml brain-derived neurotrophic factor A medium-based method for realizing the differentiation of dental tissue stem cells into nerve cells according to Item 1.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0012] The components shown in the figures are respectively assigned the following reference numbers: A. Axon S. Cell body GC. Growth cone.

[0013] Within the scope of the present invention, research has been conducted on cell applications used for the treatment of neurodegenerative diseases and the development of related drugs, the study of cell differentiation in the laboratory, and the development of drugs for the treatment of cancer types such as neuroblastoma. Therefore, in order to specifically induce the differentiation of stem cells obtained from dental pulp into dopamine neurons, a method using a novel medium has been developed within the scope of the present invention (medium-based method).

[0014] In the application of differentiation from stem cells to neurons, considering that dental pulp tissue stem cells are close to neural tissue in terms of ontogenetic origin and that the transformation of adipose (cartilage) and bone marrow-derived mesenchymal stem cells used in the field of neurogenic differentiation into neurons requires a longer time, dental pulp tissue stem cells may have higher utility compared to the application methods known in the art.

[0015] The method for neurogenic differentiation of dental pulp tissue stem cells carried out within the scope of the present invention includes the following steps: - Seed dental pulp tissue stem cells at a concentration of 5000 cells / cm 2 and, - After culturing for 24 hours, introduce the cells into the first neurogenic induction medium and continue medium application for 4 days, - Then, add the cells to the second neurogenic induction medium and continue medium application for 2 days, - Terminate differentiation after 6 days.

[0016] The compositions of the first and second neurogenic induction media expressed by the above method are as follows. First neurogenic induction medium: · Dmem / F12 Glutamax added · B-27 supplement 1% · 3-Isobutyl-1-methylxanthine (IBMX) 100 μM · Sodium valproate (VPA) 2 mM · Forskolin 0.1 μM · Basic fibroblast growth factor (bFGF) 20 ng / ml · Epidermal growth factor (EGF) 20 ng / ml Second neural induction medium: · DMEM / F12 supplemented with Glutamax · 1% B-27 supplement · 100 μM 3-isobutyl-1-methylxanthine (IBMX) · 2 mM sodium valproate (VPA) · 0.1 μM forskolin · 20 ng / ml basic fibroblast growth factor (bFGF) · 20 ng / ml epidermal growth factor (EGF) · 30 ng / ml brain-derived neurotrophic factor

[0017] The advantages provided by the present invention are listed below: · Mesenchymal stem cells can effectively differentiate into nerve cells instead of induced pluripotent stem cells. · Nerve cell differentiation can be obtained in a short period compared to other differentiation media and protocols. · Nerve cells can be formed more efficiently compared to other differentiation media and protocols. · The nerve cell differentiation of cells differentiated in other media is reversible, while the cells differentiated within the scope of the present invention show terminal transformation. · As shown in the figure, the cell cycle stops on the second day, which is necessary for effective differentiation. · There is no toxicity to cells compared to other media. · These nerve cells can be used for tissue regeneration and transplantation, and also contribute greatly to neuroscience research.

Example

[0018] Test research Cell cycle assay To observe the changes in the cell cycle, dental stem cells treated with neurogenic media were analyzed by flow cytometry. In the cell cycle assay, cells fixed on the 2nd, 4th, and 6th days of the neurogenic differentiation process were treated with RNase A and Nonidet P40, stained with propidium iodide, and analyzed.

[0019] Real-time polymerase chain reaction To observe the gene-level changes in cells treated with neurogenic media, a real-time polymerase chain reaction assay was performed. These changes are at both the morphological level and the gene expression level. The primers used were designed using Primer BLAST software (The National Center for Biotechnology = NCBI). Total RNA was isolated from gel-bound cells and DNA was synthesized. The synthesized cDNA was mixed with primers and a Fermentas Maxima SYBR Green mixed product to a final volume of 20 μl, and the gene expression level was analyzed using a BIO-RAD device.

[0020] Morphological analysis of differentiated cells On the final day of the differentiation process of cells treated with neurogenic media, morphological observation of the cells was performed using an optical microscope. While analyzing the differentiated cells, the development and presence of cells and structures characteristic of nerve cells were morphologically examined.

[0021] Cresyl violet staining of differentiated cells On the final day of the differentiation process of cells treated with neurogenic media, staining of Nissl bodies specific to nerve cells found in the cells was performed. It was confirmed that the cresyl violet staining solution applied to the cells stained the ribosomes of the granular vesicles found in the cell bodies (S) of nerve cells and showed a dark blue-violet color. On the other hand, undifferentiated dental tissue stem cells may be detected in a light pink color.

[0022] Literature [1]. Huang, G.T., S. Gronthos, and S. Shi, Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res, 2009. 88(9): p. 792-806. [2]. Niibe, K., et al., The potential of enriched mesenchymal stem cells with neural crest cell phenotypes as a cell source for regenerative dentistry. Jpn Dent Sci Rev, 2017. 53(2): p. 25-33. [3]. Goldman, S.A., Stem and Progenitor Cell-Based Therapy of the Central Nervous System: Hopes, Hype, and Wishful Thinking. Cell Stem Cell, 2016. 18(2): p. 174-88. [4]. Qu, J. and H. Zhang, Roles of Mesenchymal Stem Cells in Spinal Cord Injury. Stem Cells Int, 2017. 2017: p. 5251313. [5]. Passier, R. and C. Mummery, Origin and use of embryonic and adult stem cells in differentiation and tissue repair. Cardiovasc Res, 2003. 58(2): p. 324-35. [6]. Ruiz-Lozano, P. and P. Rajan, Stem cells as in vitro models of disease. Curr Stem Cell Res Ther, 2007. 2(4): p. 280-92.

Claims

【Claim 1】 A medium-based method for differentiating dental tissue stem cells, which can differentiate mesenchymal stem cells obtained from dental tissue into dopaminergic neurons, the method comprising: - seeding the dental tissue stem cells at a concentration of 5000 cells / cm ２ ; and - After culturing for 24 hours, adding the cells to a first neurogenic induction medium and continuing medium application for 4 days; - Then, adding the cells to a second neurogenic induction medium and continuing medium application for 2 days; - Ending differentiation after 6 days characterized by comprising, wherein the first neurogenic induction medium comprises: - Dmem / F12 with Glutamax added; - 1% B-27 supplement; - 100 μM 3-isobutyl-1-methylxanthine (IBMX); - 2 mM sodium valproate (VPA); - 0.1 μM forskolin; - 20 ng / ml basic fibroblast growth factor (bFGF); - 20 ng / ml epidermal growth factor (EGF) and the second neurogenic induction medium comprises: - Dmem / F12 with Glutamax added; - 1% B-27 supplement; - 100 μM 3-isobutyl-1-methylxanthine (IBMX); - 2 mM sodium valproate (VPA); - 0.1 μM forskolin; - 20 ng / ml basic fibroblast growth factor (bFGF); - 20 ng / ml epidermal growth factor (EGF); - 30 ng / ml brain-derived neurotrophic factor and a method.

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