A culture-based method for achieving neuronal differentiation of dental stem cells

Abstract

The present invention relates to the use of a culture medium-based method for inducing the specific differentiation of dental stem cells into dopaminergic neurons. The purpose of the present invention is to develop cell applications for treating degenerative neurological diseases and drugs related to the diseases.

Description

Field of the Invention

[0001] The present invention relates to use of a culture medium-based method for inducing specific differentiation of dental stem cells into dopaminergic neurons. Background of the Invention

[0003] One source of mesenchymal stem cells in the human body is dental tissue. Stem cells isolated from different parts of the tooth have been characterized. Examples of these are cells isolated from dental pulp, periodontal connective tissue, and unerupted impacted teeth [1]. Dental derived stem cells (DDSCs) are attractive because they are derived from neuroectodermal cells (neural crest cells) that form from the ectoderm of the neural tube during the embryonic period [2]. These cells have the ability to differentiate into many different types of cells and tissues. In addition to odontogenesis, these cells have also been found to have the ability to undergo osteogenesis, adipogenesis, chondrogenesis, and neurogenesis [1]. Because they are derived from neural crest cells and exhibit some neural-specific expression profiles, DDSCs are considered a promising cell type for the treatment of neurodegenerative diseases. The incidence of neurodegenerative diseases is increasing, and the occurrence of neurodegenerative diseases is caused by the continuous and irreversible loss of function or death of nerve cells. Factors such as the limited formation of new neurons in the central nervous system, the presence of factors that delay recovery, and the structure of the skull that makes surgical intervention difficult make these diseases difficult to treat.

[0004] One of the important approaches for treating diseases of this type caused by damage or loss of nerve cells is the use of stem cells [3]. It has been observed that after being applied to a defined location with appropriate techniques, stem cells are able to migrate to the damaged area, have the ability to differentiate, and contribute to the damage repair process by stimulating endogenous stem cells [4]. Tissues such as teeth, bone marrow, blood, cartilage, and fat can now serve as sources of adult stem cells [5]. The ability of these types of stem cells to differentiate into cell types lost in neurodegenerative diseases will make it possible to create individual-specific disease models in a laboratory setting using cells prepared from tissues of the central nervous system that can be more easily taken from patients [6]. Studying the neurogenic differentiation capacity of these different types of stem cells will enable the identification of cell types and methods that can make the greatest contribution to the replacement of dead nerve cells in the treatment of neurodegenerative diseases, as well as to the development of various therapies by creating individual-specific cell culture models of these diseases.

[0005] The differentiation of induced pluripotent stem cells into neurons has the following disadvantages, such as low differentiation yield in culture, high mutation rate, higher cost than culturing and differentiating mesenchymal cells, and the source cells contain viral genes and therefore have a considerable potential to cause carcinogenicity in in vivo cell transfer. In addition, due to the fact that dental stem cells are closer to neural tissue in embryological origin, the time period spent by adipose and bone marrow-derived mesenchymal stem cells known for use in neurogenic differentiation is longer and does not have the same high potential as dental stem cells. When obtaining mesenchymal stem cells, the following disadvantages occur, such as methodological difficulties in obtaining cells (particularly from bone marrow and cartilage), and the inability to obtain a large number of cells. Finally, in the differentiation of stem cells from different sources, substances such as DMSO, BHA or β-mercaptoethanol, which destroy the cell morphology and cause pseudo-neurogenesis in the cells, are used in the culture medium, making it impossible to continue culturing differentiated cells.

[0006] In an application known in the art, Chinese patent document number CN104726406 discloses a method for inducing the differentiation of dental pulp mesenchymal stem cells into neural cells. In our patent application, as described in the methods section and shown in Figure 1, during the differentiation of stem cells into neurons, when the cell density is between 40-50%, additional chemicals (VPA and IBMX) are added to arrest the cell cycle. This results in a healthy differentiation of the cells and their induction into a neural lineage, not due to high density but due to the method used.

[0007] In the application known in the art, Chinese patent document number CN1590537 discloses a method for isolating and culturing ectomesenchymal stem cells. Within the scope of this invention, the method can be used for bone tissue engineering, muscle tissue engineering, tooth tissue engineering and repairing peripheral nerve cells. In addition, in the patent application, a differentiation process of the glial lineage rather than the neural lineage is used to ensure the regeneration of peripheral nerve cells. In addition, it is different from our patent application in terms of methodology because only forskolin chemicals are used and it consists of a single step. As known in the literature, it is not enough to use cyclic AMP activators, such as forskolin alone, in differentiation. At the same time, the reliability of the differentiation method in our patent application was demonstrated by both morphology and by using cresyl violet staining ( Figure 2 -3). For all the reasons explained above, it is observed that the method in our patent application is more effective.

[0008] One of the prior art applications, U.S. patent application document number US2016296669 discloses a method for producing a transplant material for treating nerve damage. The method of the invention includes the step of culturing dental pulp stem cells in a culture medium that does not contain growth factors other than FGF2 (or bFGF (basic fibroblast growth factor)). In addition, it is known that the FGF2 factor used in the patent document only activates genes associated with the neural lineage of dental pulp-derived stem cells. However, many signaling pathways, including the cell cycle, are known to play a key role in the differentiation of stem cells. Therefore, it is clear that administering only the FGF2 factor to stem cells does not provide effective neural cell conversion. Taking this into account, it has been shown that the invention of our patent application has mature and functional neuronal gene expression as shown in Figure 4, which is due to following a scheme consisting of two steps to activate the differentiation pathway and mature the neural cells, as also described in the method. Summary of the Invention

[0009] The present invention is directed to the culture-based differentiation of dental stem cells into dopaminergic neurons.

[0010] Another object of the present invention is to develop cell applications for treating degenerative neurological diseases and drugs related to said diseases. Detailed Description of the Invention

[0012] The "Medium-Based Method for Differentiation of Dental Stem Cells into Neurons" developed to achieve the object of the present invention is illustrated schematically in the accompanying drawings, wherein:

[0013] Figure 1: Graphs showing the effects of a neurogenic induction medium created within the scope of the present invention on the cell cycle of cells depending on the number of days. [(a) Cell cycle diagram of cells treated with neurogenic induction medium for 0, 2, 4, and 6 days ((a-1), (a-2), (a-3), and (a-4))), (b) Graph showing the percentage of cells in the G0 / G1 phase, (c) Graph showing the percentage of cells in the S phase, (d) Graph showing the percentage of cells in the G2 / M phase]

[0014] Figure 2 : Shows morphological examination of cells treated with neurogenic induction medium for 6 days within the scope of the present invention; microscopic images of neuron-specific cell soma, extended axons and growth cones in the cells.

[0015] Figure 3 shows light microscopic images of cells treated with neurogenic induction medium for 6 days within the scope of the present invention and control cells grown in medium alone, after staining with neurogenic cell-specific cresyl violet dye. [(a) Neurogenic medium, (b) Control]

[0016] FIG4 is a graph showing the results of measuring neurogenic cell-specific genes in cells treated with neurogenic induction medium for 6 days within the scope of the present invention and control cells grown in the medium alone [(a) NeuN gene, (b) Nurr 1 gene, (c) DAT gene, (d) Snap 25 gene, (e) NF-H gene, (f) Map2 gene, (g) TH gene, and (h) Bcl-2 gene].

[0017] The components shown in the drawings are denoted by the following reference numerals:

[0018] A. Axon

[0019] S. cell body

[0020] GC. Growth cone.

[0021] The present invention involves research into cell applications for the treatment of neurodegenerative diseases and the development of drugs related to these diseases, as well as laboratory cell differentiation studies for the development of drugs for the treatment of cancer types such as neuroblastoma. Thus, the present invention has developed a novel culture medium-based application for specifically inducing the differentiation of dental stem cells into dopaminergic neurons.

[0022] In the application of stem cell differentiation into neurons, considering that dental stem cells are closer to neural tissue in terms of embryological origin, and that the time period taken for the conversion of adipose (cartilage) and bone marrow-derived mesenchymal stem cells into neurons used in the prior art is longer, it can be seen that dental stem cells have a higher potential ability to differentiate into neurons compared with the applications known in the art.

[0023] The method for neurogenic differentiation of dental stem cells carried out within the scope of the present invention comprises the following steps:

[0024] - At 5000 cells / cm 2 Dental stem cells were seeded at a concentration of

[0025] - After incubation for 24 hours, the cells were introduced into the first part of the neurogenic induction medium and cultured with the medium for another 4 days.

[0026] - Subsequently, the cells were introduced into the second part of the neurogenic induction medium and cultured in the medium for 2 days.

[0027] - At the end of 6 days, differentiation was terminated.

[0028] The contents of the first and second parts of the neurogenic induction medium described in the above method are as follows:

[0029] Neurogenic Induction Medium Part 1:

[0030] ● DMEM / F12 supplement for Glutamax

[0031] ● B-27 supplement%1

[0032] ● 3-Isobutyl-1-methylxanthine (IBMX) 100 µM

[0033] ● Valproic acid sodium salt (VPA) 2 mM

[0034] ● Forskolin 0.1 µM

[0035] ● Basic fibroblast growth factor (bFGF) 20 ng / ml

[0036] ● Epidermal growth factor (EGF) 20 ng / ml.

[0037] Neurogenic Induction Medium Part 2:

[0038] ● DMEM / F12 supplement for Glutamax

[0039] ● B-27 supplement%1

[0040] ● 3-Isobutyl-1-methylxanthine (IBMX) 100 µM

[0041] ● Valproic acid sodium salt (VPA) 2 mM

[0042] ● Forskolin 0.1 µM

[0043] ● Basic fibroblast growth factor (bFGF) 20 ng / ml

[0044] ● Epidermal growth factor (EGF) 20 ng / ml

[0045] ● Brain-derived neurotrophic factor 30 ng / ml.

[0046] The advantages provided by the present invention can be listed as follows:

[0047] ● Provides efficient neuronal differentiation in mesenchymal stem cells rather than induced pluripotent stem cells.

[0048] ● Neuronal differentiation is observed in a shorter time period compared to other differentiation media and protocols.

[0049] ● More efficient neuronal formation was observed compared to other differentiation media and protocols.

[0050] • Neuronal differentiation of cells differentiated by other media is reversible, whereas cells differentiated within the scope of the present invention exhibit terminal transformation.

[0051] ● Also as shown in the figure, the cell cycle arrests on day 2, which is required for efficient differentiation.

[0052] ● Did not cause any toxicity to cells when compared to other culture media.

[0053] ● These neuronal cells can be used for tissue regeneration and transplantation, and they also make great contributions to neuroscience research.

[0054] Experimental studies

[0055] Cell cycle assay

[0056] To observe changes in cell cycle phase, flow cytometry analysis was performed on dental stem cells treated with neurogenic medium. For cell cycle analysis, cells fixed on days 2, 4, and 6 of neurogenic differentiation were analyzed by RNase A and Nonidet P40 treatment and propidium iodide staining.

[0057] Real-time polymerase chain reaction

[0058] Real-time polymerase chain reaction was performed to observe the changes in gene expression in cells treated with neurogenic culture medium. These changes were at the morphological and gene expression levels. The primers used were designed using Primer BLAST software (National Center for Biotechnology, USA = NCBI). Total RNA was isolated from the cells, gel-assembled and synthesized into cDNA. In a Fermentas Maxima SYBR Green mixed product, the synthesized cDNA was mixed with primers to a final volume of 20 μl, and the expression level of the gene was analyzed using a BIO-RAD device.

[0059] Morphological analysis of differentiated cells

[0060] On the final day of differentiation, cells treated with neurogenic medium were analyzed morphologically under a light microscope. The development and presence of characteristic cellular and neuronal structures were examined morphologically during the analysis of differentiated cells.

[0061] Cresyl violet staining of differentiated cells

[0062] On the final day of differentiation, cells treated with neurogenic medium were stained for the characteristic Nissl bodies found in neuron-specific cells. Cresyl violet dye applied to the cells stains the ribosomes of the granular endoplasmic reticulum found in the somata (S) of neural cells, resulting in a deep bluish-purple color. On the other hand, undifferentiated dental stem cells were detected in a pale pink color.

[0063] References

[0064]

Claims

1. A culture-based method for achieving the differentiation of dental stem cells into neurons, wherein the method is capable of differentiating mesenchymal stem cells obtained from dental tissue into dopaminergic neurons, and comprises the following steps: - At 5000 cells / cm 2 Dental stem cells were seeded at a concentration of - After incubation for 24 hours, the cells were introduced into the first part of the neurogenic induction medium and cultured with the medium for another 4 days, - Subsequently, the cells are introduced into the second part of the neurogenic induction medium and cultured with the medium for 2 days. - At the end of 6 days, differentiation was terminated with the first portion of neurogenic induction medium containing: - DMEM / F12 supplemented with Glutamax, -B-27 supplement 1%, -3-isobutyl-1-methylxanthine (IBMX) 100 μM, -Valproic acid (VPA) sodium salt 2mM, -Forskolin 0.1 μM, - basic fibroblast growth factor (bFGF) 20 ng / ml, - Epidermal growth factor (EGF) 20 ng / ml, and wherein the second part of the neurogenic induction medium contains: - DMEM / F12 supplemented with Glutamax, -B-27 supplement 1%, -3-isobutyl-1-methylxanthine (IBMX) 100 μM, -Valproic acid (VPA) sodium salt 2mM, -Forskolin 0.1 μM, - basic fibroblast growth factor (bFGF) 20 ng / ml, - Epidermal growth factor (EGF) 20ng / ml, - Brain-derived neurotrophic factor 30ng / ml.

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