Pharmaceutical composition for treating demyelinating disease, containing supernatant of immortalized mesenchymal stem cells, and pharmaceutical preparation containing said composition as active ingredient

Abstract

The purpose of the present invention is to provide a therapeutic agent for demyelination diseases, which comprises, as an active ingredient, a culture supernatant of immortalized mesenchymal stem cells into which a specific gene has been inserted. The present invention provides a therapeutic agent for demyelinating diseases, which contains, as an active ingredient, a culture supernatant of immortalized mesenchymal stem cells into which either an hTERT gene or a pTERT gene, and a bmi-1 gene, an HPV-E6 gene, or an HPV-E7 gene are inserted. The mesenchymal stem cells are preferably derived from a human or a pig, and the culture supernatant preferably contains a component that acts on the receptor c-MET of a hepatocyte growth factor (HGF), said component being obtained by culturing the mesenchymal stem cells for 24-96 hours.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating demyelinating diseases comprising a culture supernatant of immortalized mesenchymal stem cells, and a pharmaceutical preparation containing the composition as an active ingredient. More specifically, it relates to a pharmaceutical composition for treating demyelinating diseases caused by infection or autoimmune reaction, such as Guillain-Barré syndrome, or demyelinating diseases caused as a side effect of treatment of other diseases such as chemotherapy, and a pharmaceutical preparation containing the composition as an active ingredient. Background Art

[0002] In recent years, cell therapy using mesenchymal stem cells (hereinafter sometimes referred to as "MSC") has been actively studied. Here, "cell therapy" is a method of using cells themselves as a therapeutic means, and can be defined as a method that aims to cure various diseases using one's own cells (autologous) or non-self cells (third party, i.e., allogeneic). With the progress of cell / genetic engineering technology in recent years, various cell therapies have been developed, such as: (1) a method of directly using the administered cells to treat a disease; (2) a method of using the administered cells to produce therapeutic cells in vivo and using these cells for treatment; (3) a cell therapy that artificially processes cells removed from a living body to exert a therapeutic effect; and so on. As (1), blood transfusion therapy can be cited, and as (2), bone marrow transplantation can be cited. In addition, as (3), CAR-T cell therapy or iPS cell therapy can be cited.

[0003] It is also known that when MSC is applied to the damaged part of a certain tissue, the applied cells secrete various cytokines. And it is known that the repair or immunosuppression of damaged tissue is produced by the paracrine effect of the secreted cytokines, etc. On the other hand, if the cells are removed from the cell culture supernatant (conditional medium: Conditioned Medium, hereinafter sometimes referred to as "CM") of MSC, there is no need to consider the immune rejection caused by tissue compatibility antigens, and as described below, the safety is also high, so it has attracted people's attention (non-patent document 1, hereinafter referred to as "prior art 1").

[0004] Nervous tissue can be roughly divided into the central nervous system consisting of the brain and spinal cord, and the peripheral nervous system extending from the brain and spinal cord to the periphery. The neural network consists of axons and dendrites extending from the cell bodies of nerve cells. The axons are covered by an insulating structure called myelin sheath, which enables rapid information transmission to distant cells. The cells that make up the myelin sheath are called oligodendrocytes in the central nervous system and Schwann cells in the peripheral nerves.

[0005] The above-mentioned myelin sheath may degenerate or fall off due to various reasons sometimes. The diseases caused by the degeneration or falling off of myelin sheath are collectively referred to as demyelinating diseases. The causes of demyelinating diseases are various, and known to have immunity, heredity, ischemia, infection, drug-induced, etc. As central nervous system demyelinating diseases, for example, multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, atopic myelitis, progressive multifocal leukoencephalopathy, HTLB-1 related myelopathy, HIV-related leukoencephalopathy, subacute sclerosing panencephalitis, osmotic demyelinating syndrome, drug-induced leukoencephalopathy, etc. can be enumerated. As peripheral nervous system demyelinating diseases, for example, Guillain-Barré syndrome, Fisher syndrome, diabetic neuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, multifocal motor neuropathy and herpes zoster, etc. can be enumerated.

[0006] As the cause of the above-mentioned immune demyelinating diseases, autoimmune abnormalities are well known. The organism is equipped with an immune system as a biological defense mechanism. When the immune system functions normally, it attacks foreign matter (substances recognized as non-self) and eliminates it, but does not react to antigens of self-tissues and does not attack self-tissues. However, if the normal function of the immune system is impaired, and the induction of T cells that recognize self-tissues as foreign matter or antibodies called autoantibodies is produced, the immune cells will attack specific cells or tissues as targets, and an immune response targeting the self will occur. It is known that when such an autoimmune reaction occurs in neural tissue, a neuropathy called autoimmune neuritis (hereinafter sometimes referred to as "EAN") will occur. In particular, immune abnormalities that attack one's own myelin can lead to the occurrence of demyelinating diseases.

[0007] Examples of demyelinating diseases caused by EAN include multiple sclerosis, acutely progressive Guillain-Barré syndrome and Fisher syndrome, chronically progressive chronic inflammatory demyelinating polyradiculoneuropathy (hereinafter sometimes referred to as "CIDP"), MAG antibody-positive neuropathy, and multifocal motor neuropathy (hereinafter sometimes referred to as "MMN"), all of which are accompanied by peripheral neuropathy.

[0008] Among the above diseases, Guillain-Barré syndrome produces symptoms such as numbness and weakness in the extremities of the hands and feet. After the onset of such symptoms, the symptoms progress rapidly within a few days to two weeks, and the symptoms peak 2 to 4 weeks after the onset of neurological symptoms. It is known that when the disease becomes severe, the paralysis of the limbs worsens, and walking requires assistance. In addition, in more than 10% of patients, the respiratory muscles are paralyzed, requiring artificial respirators. About half of the patients develop cranial neuropathies such as facial nerve paralysis, diplopia, and dysphagia. The cause of the severe disease is said to be axonal degeneration related to demyelination, and early treatment is considered important.

[0009] It is said that 60% of Guillain-Barré syndrome is caused by the production of anti-ganglioside antibodies. Therefore, in order to weaken the effect of anti-ganglioside antibodies, intravenous immunoglobulin therapy and plasma purification therapy (Non-patent Document 2, hereinafter referred to as "Prior Art 2") are implemented. However, there are still patients who are ineffective using such treatments. In this regard, in addition to the above-mentioned antibodies, cells related to cellular immunity, such as T cells, are also considered to be the cause, but the details are still unclear.

[0010] Peripheral neuropathy is a series of symptoms caused by damage to the nerves (peripheral nerves) that extend from the brain and spinal cord to the periphery. It is a disease that causes numbness and pain in the hands and feet due to various reasons. It is known to be caused by diseases such as infections and diabetes, and can also be caused by side effects of various drugs.

[0011] As examples of such drugs, hyperlipidemia treatment drugs, antiviral drugs, anti-tuberculosis drugs, etc. can be cited, and cancer treatment drugs are particularly problematic. It is said that half of the population in Japan suffers from cancer recently, and cancer accounts for 1 / 3 of the causes of death. It is known that peripheral neuropathy is observed in most patients during cancer treatment. That is, in cancer treatment, chemotherapeutic agents are often used, but it is known that when using platinum-based preparations such as cisplatin, carboplatin, oxaliplatin, and nedaplatin, vinca alkaloid preparations such as vincristine, vinblastine, and vinorelbine, taxane preparations such as paclitaxel and docetaxel, cytarabine as a metabolic antagonist, ifosfamide as an alkylating agent, etc., peripheral neuropathy is likely to occur as a side effect.

[0012] For example, the above-mentioned vinca alkaloids and taxanes act on microtubules in cancer cells, but are believed to damage microtubules of nerve cells at the same time, causing neurological symptoms. In addition, the above-mentioned platinum-based drugs are believed to directly damage nerve cells and cause pathological changes in the axons of nerve cells.

[0013] In addition, cancer treatment drugs such as amiodarone hydrochloride, interferon-α, and tacrolimus are said to cause peripheral neuropathy by damaging Schwann cells covering axons. In this case, axons and cell bodies are mostly relatively normal, and good recovery is expected by stopping the drug administration early, but if the lesion progresses, the axons undergo secondary degeneration and sometimes recovery is not expected.

[0014] The above-mentioned peripheral neuropathy is a common side effect of chemotherapy for cancer treatment and is known as chemotherapy-induced peripheral neuropathy (hereinafter sometimes referred to as "CIPN"). The structure of nerves, nerve cells, and their degeneration are described below.

[0015] In the case of CIPN mentioned above, relief of peripheral nerve symptoms such as numbness of hands and feet has been achieved by temporarily stopping anticancer drugs, co-administering calcium / magnesium, administering Chinese herbal medicines or vitamin preparations (vitamin B6, vitamin B12, multivitamin preparations, etc.) (Non-patent document 3, hereinafter referred to as prior art 3).

[0016] Prior art literature

[0017] Non-patent literature

[0018] Non-patent literature 1: Vizoso FJ, Eiro N, Cid S, Schneider J, Perez-Fernandez R. Mesenchymal Stem Cell Secretome: Toward Cell-Free Therapeutic Strategies in Regenerative Medicine. Int J Mol Sci. 2017 Aug 25; 18(9): 1852. doi: 10.3390 / ijms18091852.

[0019] Non-patent document 2: Dimachkie MM, Barohn RJ. Guillain-Barre syndrome and variants. Neurol Clin. 2013 May; 31(2): 491-510. doi: 10.1016 / j.ncl.2013.01.005.

[0020] Non-patent literature 3: Tsai CH, Lin YH, Li YS, Ho TL, Hoai Thuong LH, Liu YH. Integrated Medicine for Chemotherapy-Induced Peripheral Neuropathy. Int J Mol Sci. 2021 Aug 26; 22(17): 9257. doi: 10.3390 / ijms22179257. Summary of the invention

[0021] Problems to be solved by the invention

[0022] Prior art 1 uses CM of MSCs that do not contain cells, so there is no need to consider immune rejection, and the ethical restrictions that are problematic in cell therapy, as well as the possibility of worrying embolism or tumor formation are low. From these aspects, it is highly safe and is an excellent technology. However, in the case of culturing non-immortalized MSCs, due to cell aging, the proliferation ability decreases at a maximum of about 10 generations. Therefore, it is necessary to prepare MSCs from the donor again, and it is difficult to perform quality control of continuously supplying CM containing stable components.

[0023] Prior art 2 is a standard method used in the treatment of the above-mentioned Guillain-Barré syndrome, including intravenous immunoglobulin therapy (hereinafter sometimes referred to as "IVIg") and plasma purification therapy. These treatments are implemented to weaken the effects of anti-ganglioside antibodies. However, there are still patients who do not respond to these treatments, and the reason is still unclear. Therefore, there is a strong social demand for new therapeutic drugs that are more effective than such methods for demyelinating diseases including Guillain-Barré syndrome.

[0024] In addition, as mentioned above, anti-cancer chemotherapy is a treatment method for most cancer patients, and the dosage can be increased to the maximum tolerated dose. However, when the dosage of the drug increases, CIPN as its side effect will frequently occur. Prior art 3 can alleviate the symptoms of CIPN to a certain extent, and is an excellent method in this respect. However, since it is a symptomatic treatment, when the symptoms cannot be successfully alleviated, it is sometimes necessary to reduce the dosage, stop the drug or terminate the administration, which will lead to the problem of cancer symptom progression. In addition, when CIPN is not alleviated, it will also bring about the problem of reduced QOL of the patient.

[0025] In summary, there is a strong social demand for a pharmaceutical composition that can be used for highly safe cell-free therapy and can fundamentally treat demyelinating diseases, and a pharmaceutical preparation containing such a composition as an active ingredient.

[0026] Means for solving problems

[0027] The inventors of the present invention have conducted intensive research under the above circumstances and have completed the present invention. The purpose of the present invention is to provide a pharmaceutical composition and a pharmaceutical preparation for demyelinating diseases that contain a culture supernatant of immortalized mesenchymal stem cells into which a specific gene is inserted as an active ingredient and can be used to treat demyelinating diseases.

[0028] The first aspect of the present invention relates to a composition capable of inducing the regeneration of myelin sheaths or the proliferation of Schwann cells, which is a composition comprising a culture supernatant of immortalized mesenchymal stem cells into which a combination of two or three genes selected from the group consisting of Bmi-1 gene, HPV16-E6 gene and HPV16-E7 gene and telomerase reverse transcriptase (TERT) gene has been introduced. Here, the regeneration of myelin sheaths refers to the re-myelination of axons exposed due to the shedding of Schwann cells through the Schwann cell proliferation effect of the present invention, or the restoration of thinned myelin sheaths to their original thickness.

[0029] In addition, the combination of the above genes is preferably Bmi-1 gene, HPV16-E6 gene, HPV16-E7 gene and TERT gene.

[0030] Here, the mesenchymal stem cells are preferably dental pulp stem cells, and the dental pulp stem cells are preferably human dental pulp stem cells. In addition, the composition is preferably a culture supernatant obtained by introducing the gene into the stem cells using a viral vector for immortalization, and culturing the immortalized stem cells obtained in a serum-free medium for 24 to 96 hours, preferably 48 to 80 hours. More preferably, the culture time is about 72 hours.

[0031] In addition, the composition preferably contains at least a component that acts on c-MET, a receptor for hepatocyte growth factor (HGF), and more preferably, the component is HGF.

[0032] Another aspect of the present invention relates to a pharmaceutical preparation for treating a demyelinating disease, comprising the above composition as an active ingredient. Here, when the total weight of the preparation is set to 100%, the above composition is preferably contained in an amount of 10 to 90% by weight. More preferably, the above composition is contained in an amount of 25 to 75% by weight.

[0033] Preferably, the demyelinating disease is a central nervous system disease and is any lesion selected from the group consisting of multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, atopic myelitis, progressive multifocal leukoencephalopathy, HTLB-1-related myelopathy, HIV-related leukoencephalopathy, subacute sclerosing panencephalitis, osmotic demyelination syndrome and drug-induced leukoencephalopathy.

[0034] Preferably, the demyelinating disease is a peripheral neuropathy, and is any one selected from the group consisting of Guillain-Barré syndrome, Fisher's syndrome, diabetic neuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, multifocal motor neuropathy, chemotherapeutic agent-induced peripheral neuropathy, and herpes zoster.

[0035] The therapeutic preparation is preferably in any dosage form selected from the group consisting of powder, tablet, capsule, liquid, injection and patch.

[0036] The second aspect of the present invention relates to a pharmaceutical preparation for treating chemotherapeutic agent-induced peripheral neuropathy, which comprises, as an active ingredient, a composition comprising a culture supernatant of immortalized mesenchymal stem cells into which a combination of two or three genes selected from the group consisting of Bmi-1 gene, HPV16-E6 gene, and HPV16-E7 gene and telomerase reverse transcriptase (TERT) gene are inserted. Here, the chemotherapeutic agent is preferably an anticancer agent, more preferably a vinca alkaloid preparation or a taxane preparation.

[0037] Effects of the Invention

[0038] The therapeutic agent for demyelinating diseases according to the present invention can promote the alleviation or rapid recovery of symptoms of demyelinating diseases, peripheral neuropathy, especially Guillain-Barré syndrome and CIPN by using the culture supernatant of immortalized mesenchymal stem cells into which Bmi-1, HPV-E6, HPV-E7 and TERT genes are inserted as an active ingredient.

[0039] Furthermore, the therapeutic composition for a demyelinating disease of the present invention can promote the proliferation of Schwann cells, thereby recovering from the reduction in nerve function. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The diagrams show the myelin sheath and neurotransmission status of normal, Guillain-Barré syndrome, and chemotherapy-induced peripheral neuropathy nerve cells. (A) shows the myelin sheath and neurotransmission status of normal nerve cells. (B) shows the myelin sheath and neurotransmission status of nerve cells with Guillain-Barré syndrome. (C) shows the myelin sheath and neurotransmission status of nerve cells with chemotherapy-induced peripheral neuropathy.

[0041] Figure 2 This is a diagram schematically showing the relationship between the development of peripheral neuropathy and the immune system in patients with Guillain-Barré syndrome.

[0042] Figure 3 The graphs show the method for preparing mice with autoimmune neuritis and the changes in clinical scores when SHED-CM was intraperitoneally administered to the mice with autoimmune neuritis. Figure 3 (A) shows the dosing schedule when P0 (180-199) was administered to C57BL / 6 mice to create autoimmune neuritis model mice and then DMEM or SHED-CM was administered. Figure 3 (B) shows the use of the above Figure 3 (A) Changes in clinical sign scores during treatment with the dosing regimens.

[0043] Figure 4 Show Figure 3 Autoimmune neuritis mice were intraperitoneally administered with DMEM ( Figure 4 (A)) or SHED-CM( Figure 4 (B) Optical microscope image of toluidine blue staining of nerve cell axons and myelin sheaths ( Figure 4 (A1, A2) and Figure 4 (B1, B2)), and electron microscope images ( Figure 4 (A3) and (B3)). The black bars in the electron microscopy images are scale bars (10 μm).

[0044] Figure 5 represents the results of expression analysis by quantitative PCR. Figure 5 (A) is a graph in which the vertical axis represents the relative ratio when the expression level of myelin basic protein (Myelin Basic Protein, hereinafter sometimes referred to as "MBP") is divided by the expression level of hypoxanthine guanine phosphoribosyltransferase (hereinafter sometimes referred to as "HPRT") as an internal standard gene. Figure 5 (B) shows the results of Western blotting after gel electrophoresis, which shows the effect of SHED-CM on the expression level of MBP or actin.

[0045] Figure 6 It is a graph showing the expression levels of TNF-α, IFN-γ, and IL-17A genes in nerve cells when SHED-CM was administered. Figure 6 (A1) to (A3) show the expression levels of the above genes in sciatic nerve cells. Figure 6 (B1) to (B3) show the expression levels of the above genes in cauda equina neurons.

[0046] Figure 7 The results of Western blotting after gel electrophoresis show that the amount of phosphorylated c-Met protein in sciatic nerve cells is increased by administration of SHED-CM.

[0047] Figure 8 The therapeutic effects of various SHED-CMs on autoimmune neuritis model mice established from wild-type (hereinafter sometimes referred to as "WT") mice are shown. Figure 8 (A) shows the preparation of autoimmune neuritis model mice using WT mice, and the administration schedule of SHED-CM to the model mice. Figure 8(B) is a graph showing that the effect of SHED-CM on the above model mice tends to be reduced by co-administration of anti-HGF antibodies.

[0048] Fig. 9 These are optical microscope images showing the state when Schwann cell line IMS32 was cultured in DMEM or DMEM medium containing SHED-CM. Fig. 9 (A) is an optical microscope image showing the shape change of IMS32 cells before the experiment. Fig. 9 (B1) is an optical microscope image showing the shape change of IMS32 cells in a control containing 10% (v / v) DMEM, Fig. 9 (B2) is an optical microscope image showing the shape change of IMS32 cells in a control containing 30% (v / v) DMEM. Fig. 9 (C1) is an optical microscope image showing the shape change of IMS32 cells of sample 1 containing 10% (v / v) SHED-CM, Fig. 9 (C2) is an optical microscope image showing the shape change of IMS32 cells of sample 2 containing 30% (v / v) SHED-CM.

[0049] Fig.10 This is a graph showing changes in IMS32 cell proliferation when cultured in a medium containing SHED-CM. Fig.10 (A) is a graph comparing the proliferation of IMS32 cells when cultured in 1 to 30% (v / v) DMEM or DMEM containing SHED-CM. Fig.10 (B) indicates that anti-HGF and its control antibody were added to Fig.10 (A) is a graph showing changes in cell proliferation when the same culture medium is used.

[0050] Fig.11 The graph shows the proliferation of cells cultured in DMEM containing 10 to 50% (v / v) SHED-CM or 0.1 to 50 ng / mL HGF in the presence or absence of fetal bovine serum (hereinafter sometimes abbreviated to "FBS"). Fig.11 (A) shows the changes in IMS32 cell proliferation in the absence of FBS. Fig.11 (B) shows changes in IMS32 cell proliferation in the presence of 1% FBS.

[0051] Fig.12 The graph shows the results of Western blotting after electrophoresis, indicating the phosphorylation of each protein (c-met, ERK1 / 2, or AKT) and the amount of total protein in IMS32 cells when stimulated with SHED-CM or HGF.

[0052] Fig.13 The graphs are Western blotting diagrams after electrophoresis showing the amounts of MBP and p75 proteins when IMS32 cells cultured in DMEM (containing 5% FBS) were stimulated with SHED-CM, HGF, NRG1, DBcAMP, or NRG1+DBcAMP.

[0053] Fig.14 This is a graph showing the therapeutic effect of SHED-CM on chemotherapy-induced peripheral neuropathy model mice created from WT mice. It shows that when 80 μg / 200 μL of paclitaxel was intraperitoneally administered every other day, SHED-CM was administered to model mice, and neuropathy was alleviated and recovered faster than the control group administered with control medium DMEM.

[0054] Fig.15 This is a table showing the pressure strength of fiber filaments and accompanying information (cited from the Dinamic Global, LLC explanatory book).

[0055] Fig.16 Table 1 shows k values ​​used in calculations of the von Frey filament test.

[0056] Fig.17 Table 2 shows the k values ​​used in the calculation of the von Frey fiber test. DETAILED DESCRIPTION

[0057] The following reference Figure 1 to Figure 17 One embodiment of the present invention will be described. It should be noted that in the following description and drawings, the same or equivalent elements are denoted by the same reference numerals, and duplicate descriptions are omitted.

[0058] The therapeutic composition using the culture supernatant (CM) of mesenchymal stem cells as the first embodiment of the present invention is prepared in the following order. That is, (S1) a combination of specific genes is inserted into human dental pulp stem cells to prepare immortalized stem cells, (S2) the immortalized stem cells obtained are cultured to prepare the culture supernatant, and (S3) a composition containing the culture supernatant as an active ingredient that can induce the regeneration of myelin sheaths or the proliferation of Schwann cells is prepared.

[0059] Here, a method using dental pulp stem cells as an example of mesenchymal stem cells is described. Dental pulp stem cells are a type of stem cells with regenerative ability contained in the dental nerve. Because they are protected by hard materials such as teeth, they are not exposed to ultraviolet rays or radiation, and therefore have the characteristic that their genes are not easily damaged.

[0060] In addition, the combination of the above genes includes bmi-1, HPV-E6, HPV-E7 and TERT. TERT is a gene for telomere repair enzymes, and bmi-1 is a gene for Bmi-1, one of the proteins that constitute the polycomb complex. Here, Bmi-1 is necessary for maintaining hematopoietic stem cells and has the effect of increasing hematopoietic stem cells by enhancing activity. E6 and E7 are early genes of DNA of HPV-16 or HPV-18.

[0061] First, the deciduous teeth are disinfected with disinfectants such as chlorhexidine and povidone-iodine solution.

[0062] The crown of the tooth is cut and the pulp tissue is recovered using a dental reamer. The collected pulp tissue is suspended in a basal culture medium, such as Dulbecco's Modified Eagle's Medium (hereinafter referred to as "DMEM") containing 5 to 15% bovine serum (hereinafter sometimes referred to as "CS") and 50 to 150 units / mL of antibiotics. Then, 1 to 5 mg / mL of collagenase and 1 to 5 mg / mL of dispase are used for treatment at 37°C for 0.5 to 2 hours.

[0063] As the above-mentioned basal medium, in addition to DMEM, Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12) (SIGMA, GIBCO, etc.), RPMI1640 medium, etc. can also be used. Two or more basal mediums can also be used in combination. As an example of a mixed medium, a medium (for example, commercially available under the trade name: IMDM / Ham F12 (GIBCO)) obtained by mixing equal amounts of IMDM and Ham F12 can be cited.

[0064] In addition, components added to the basal medium include fetal calf serum (hereinafter sometimes referred to as "FCS"), human serum, goat serum and other sera, serum substitutes (Knockout serum replacement (Knockout serum replacement, KSR) etc.), bovine serum albumin (hereinafter sometimes referred to as "BSA"), antibiotics such as penicillin and streptomycin, various vitamins, and various minerals.

[0065] The above-mentioned basal medium can also be used for the cell sorting and culture described later, and the cell culture after sorting.

[0066] After the enzyme treatment, centrifuge for 3 to 10 minutes (3,000 to 7,000 rpm) to recover dental pulp stem cells. Cells are sorted using a cell filter as needed. The sorted cells are resuspended in, for example, 3 to 6 mL of the above-mentioned basal medium and inoculated into a culture dish for adherent cell culture with a diameter of 4 to 8 cm.

[0067] Next, a culture medium, such as DMEM containing 10% FCS, is added, and then cultured at 37°C in a 5% CO2 incubator for about 2 weeks. After removing the culture medium, the cells are washed once to several times using PBS or the like. Adherent dental pulp stem cells that have formed colonies can also be recovered without removing the culture medium and washing the cells. Adherent dental pulp stem cells are treated with, for example, 0.025-0.1% trypsin and 0.3-1mM EDTA at 37°C for several minutes to be detached from the culture dish, and then the cells are recovered.

[0068] Next, the adherent cells separated as described above are cultured. For example, the dental pulp stem cells obtained as described above are seeded in a culture dish for adherent cell culture and cultured in an incubator under the conditions of 5% CO2 and 37°C.

[0069] Regarding subculture, for example, when the cells reach near confluence or confluence as observed with the naked eye, the cells are detached and recovered from the culture vessel using trypsin and EDTA as described above, and then seeded again into a culture vessel containing a culture medium.

[0070] Here, near confluence means that cells are attached to about 70% of the cell attachment surface in the culture container. For example, 1 to 8 subcultures are performed to proliferate the selected cells to a required number of cells, for example, about 1×10 7 After culturing as above, the cells are collected and stored in liquid nitrogen. Cells collected from various donors can also be stored in the form of a dental pulp stem cell bank.

[0071] Next, three or four genes are introduced into the primary culture cells obtained by the primary culture of the above-mentioned stem cells to prepare gene-introduced cells. Because immortalized stem cells with more individual doubling times can be obtained, the genes preferably introduced therein are hTERT and two or three genes selected from the group consisting of bmi-1, E6 and E7. Here, hTERT is the gene for human telomerase reverse transcriptase, and bmi-1 is a polycomb family gene related to the self-replication and differentiation regulation of stem cells. E6 and E7 are genes present in the open reading frame encoding the early genes used by human papillomavirus for self-replication.

[0072] Such gene introduction can be performed as follows.

[0073] A plasmid for inserting the target gene is prepared and inserted into a shuttle vector, such as pSuttle2, to clone the gene. The shuttle vector is used to transform Escherichia coli, and kanamycin-resistant transformants are selected. The plasmid DNA of the selected kanamycin-resistant transformants is purified, restriction enzyme sites are analyzed, and recombinants are identified.

[0074] 1. Preparation of DNA fragments and viral vector production process

[0075] 1.1 Preparation of DNA fragments for viral insertion

[0076] First, the sequence information of the vector used to insert the DNA fragment into the virus is obtained. In the present invention, from the aspect that the introduced gene is not expressed transiently, the virus used as the gene introduction vector is preferably any one selected from the group consisting of lentivirus, adenovirus, and retrovirus.

[0077] The following description uses the case of using a lentiviral plasmid vector (pLVSIN) as an example. For example, the sequence information of pLVSIN is downloaded from the Takara Bio website directory to confirm the multiple cloning site.

[0078] Next, a DNA fragment is prepared as follows. In the present invention, 2 to 4 genes are inserted into the above DNA fragment to prepare immortalized stem cells. The cells used are not particularly limited as long as they are stem cells obtained from mammals. From the perspective of being easy to obtain and being able to obtain immortalized stem cells with stable properties, it is preferred to use pig dental pulp tissue, pig adipose tissue, and human dental pulp tissue.

[0079] In addition, from the aspect of being able to produce immortalized stem cells with stable characteristics, the genes introduced here are preferably a combination of at least two genes selected from the group consisting of two early genes of papillomavirus, telomerase reverse transcriptase (TERT) and bmi. In addition, from the aspect of expression efficiency, the early genes of the papillomavirus are preferably human papillomavirus (hereinafter sometimes referred to as "HPV") E6 and human papillomavirus E7. From the aspect of gene expression efficiency, TERT preferably uses human TERT (hereinafter sometimes referred to as "hTERT").

[0080] Among the above genes, TERT is a gene that encodes an enzyme that lengthens the telomerase sequence that shortens with age. Human papillomavirus E6 and E7 are known to be early genes of human papillomavirus, and E6 reactivates TERT or degrades proteins with PDZ domains. Bmi-1 is known to be a polycomb family gene and is related to the self-replication and differentiation regulation of stem cells.

[0081] The following example illustrates the case of making a lentiviral vector and introducing three genes. In order to insert the human papillomavirus E6 gene, the human papillomavirus E7 gene, and the telomerase reverse transcriptase (TERT), a double-stranded DNA such as EcoRI / KoZal / E6 / T2A3 / E7 / T2A4 / hTERT / BamHI is synthesized according to the sequence information by a standard process (sequence number 1 in the sequence table). The obtained DNA fragment is cloned into the multiple cloning site of the above-mentioned lentiviral plasmid vector (pLVSIN-CMV Neo) by a standard process, and a lentiviral plasmid vector (E6E7T) with the above-mentioned three genes inserted can be obtained.

[0082] In the case of retrovirus, unlike the case of lentiviral vector, a vector with a single gene inserted is prepared and introduced by co-infection. For example, five immortalization genes (hTERT (sequence number 2 in the sequence list), E6 of human papillomavirus (sequence number 3 in the sequence list), E7 of human papillomavirus (sequence number 4 in the sequence list), porcine TERT (sequence number 5 in the sequence list), and hBmi-1 (sequence number 6 in the sequence list)) with Kozak sequence (gccacc) added upstream of the start codon are prepared according to conventional methods, cloned into the PmaC I-Hpa I site of pDON-5NEODNA (Takara No. 3657) according to conventional methods, and retrovirus plasmid vectors (pDON-5Neo hTERT vector, pDON-5Neo HPV16E6 vector, pDON-5Neo HPV16E7 vector, pDON-5Neo pHTERT vector, and pDON-5Neo hBmi1 vector) can be prepared.

[0083] Thereafter, the five plasmid DNAs prepared as described above are used to transform Escherichia coli according to a conventional method, and the obtained transformants are cultured in a CO 2 incubator to obtain transfection-grade plasmid DNAs.

[0084] Next, G3T-hi cells were cultured at 5.5-6.5×10 6 The cells / dish are inoculated into the desired microplate, cultured in a 5% CO2 incubator at about 37°C for about 20 to 28 hours, a desired amount of transfection reagent (e.g., 0.3 to 0.5 mL of TransIT-293) is added, 4 plasmid vectors containing any one of TERT from human or porcine are selected from the above 5 plasmid vectors, pGP and pE-Ampho (both of which are vectors included in the retrovirus packaging kit Ampho) are co-introduced, and further cultured under the same conditions for 40 to 56 hours, thereby enabling the introduction of these genes.

[0085] 1.2 Preparation of vector-producing cells

[0086] In parallel with the above operations, recombinant lentiviral vector-producing cells are prepared. As such a cell line, commercially available cell lines such as Lenti-X 293T (manufactured by Clontech, No.: 632180) can be used. Lenti-X 293T can be cultured using a culture medium containing fetal bovine serum and antibiotics. As such a culture medium, for example, minimum essential medium (MEM), DMEM, etc. can be cited. From the perspective of cell proliferation efficiency, for example, it is preferred to use DMEM (manufactured by SIGMA, St. Louis, MO) containing 5% to 15% fetal bovine serum (FBS, manufactured by HyClone), 0.5% to 2% antibiotics (penicillin / streptomycin, manufactured by Gibco), etc.

[0087] For example, DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin may be prepared and used as the basal medium. Hereinafter, in this specification, this medium is referred to as "293T basal medium".

[0088] For example, when Lenti-X 293T is used as the cell line, 1×10 5 ~5×10 5 cells / mL of cell suspension, add 10mL of the suspension to a 10cm culture dish, culture in a 5% CO2 incubator for about 24 hours, and then subculture according to the state of the cells until use. When using, first remove the culture supernatant of the cells that are continuously subcultured, wash with PBS, add a commercially available stripping agent to strip the cells from the culture dish and recover them. Next, take the desired amount of about 3 to 5 times diluted cell suspension, add trypan blue staining solution, and use a hemocytometer to count the number of live cells. Add the above-mentioned basal culture medium to prepare about 5×10 5 cells / mL of cell suspension, for example, inoculated in a culture dish at a desired concentration, and cultured under desired conditions. 6 ~4×10 6 The cells / 4 mL were seeded in a collagen-coated culture dish of 6 cm in diameter, and then cultured at about 37° C. in a 5% CO 2 incubator for about 24 hours, and the culture medium was replaced and culture was further continued.

[0089] In addition, as retrovirus preparation cells, G3T-hi cells are preferably used, which are GnT-III high-expressing cell lines (manufactured by Takara Bio Co., Ltd.) obtained by introducing human N-acetylglucosaminyl transferase III (N-acetylglucosaminyl transferase III: GnT-III) into the human kidney-derived cell line 293T (G418 resistance) using the hygromycin resistance gene.

[0090] The G3T-hi cells are designed as follows: using the retrovirus packaging kit Eco or Ampho (manufactured by Takara Bio Co., Ltd., product number 6160, 6161), the expression vector plasmids of the gag-pol and env genes and the recombinant retrovirus vector plasmids inserted with the target gene are co-introduced, thereby being able to rapidly and transiently produce high-titer recombinant viruses. And the reason is that since the G3T-hi cells are derived from 293T, the SV40 T antigen gene is introduced into them, and through the action of this gene, the RNA of the retrovirus is amplified to obtain a high-titer virus solution. Usually, by using the transient expression of the retrovirus packaging kit, a high-titer virus solution containing 10 5 ~10 7 cfu / mL of virus solution.

[0091] In the above G3T-hi cells, the cell membrane sugar chains are modified by GnT-III. It is speculated that the sugar chains of the membrane proteins of the recombinant retrovirus obtained from the above G3T-hi cells are modified by GnT-III because the budding retrovirus is entangled on the host cell membrane. Through this sugar chain modification, the affinity of the retrovirus prepared using the above G3T-hi cells for RetroNectin (recombinant human fibronectin fragment) is increased, so by using RetroNectin as a coating agent for the culture vessel, the efficiency of gene introduction into the target cells can be significantly improved. RetroNectin is particularly effective for gene introduction targeting blood cell lineage cells.

[0092] When using G3T-hi cells, from the perspective of retrovirus production efficiency, it is preferred to use a culture medium containing DMEM containing glucose (4.5 g / L) and L-glutamine (584 mg / L) supplemented with 10% FBS and 1% penicillin / streptomycin instead of the above-mentioned 293T basal medium.

[0093] 1.3 Preparation of viral vector

[0094] The following description will be given using a lentiviral vector as an example.

[0095] The lentiviral vector plasmid prepared as above is added to the culture dish with cells cultured as described above, and co-transfection is performed. Such co-transfection can use a commercially available packaging system, such as Lenti-X HTX packaging system (manufactured by Clontech). The specific process can be carried out according to the operating manual attached to such a system.

[0096] After the co-transfection is completed, the culture medium is replaced with a fresh complete medium and cultured at about 37°C for 24 to 48 hours. The titer of the viral vector is determined to determine the timing of recovery, and the culture supernatant containing the viral vector is recovered when the viral titer reaches the maximum. The titer of the viral vector can be determined using a commercially available simple titer determination kit, such as Lenti-X GoStix (manufactured by Clontech).

[0097] For example, on the next day after the culture medium is replaced, the culture supernatant of the above-mentioned culture dish is sucked out with a syringe of the desired size for recovery, and the recovered culture supernatant is filtered to obtain the viral vector. For example, the culture supernatant is sucked out with a 10mL disposable syringe (Terumo Co., Ltd.), and then a filter of, for example, 0.45 μm (MILEX-HV, Millipore Co., Ltd.) is installed on the syringe, and the culture supernatant in the syringe is filtered, and the viral vector solution is recovered in, for example, a 15mL tube.

[0098] Next, the presence of the recombinant lentiviral vector is confirmed using the reagents included in the commercially available simple titer determination kit. For example, a desired amount of viral vector solution, such as 20 μL, can be added to the above-mentioned Lenti-X GoStix S, and Chase buffer 1 can be further added, and the reaction is allowed to proceed at room temperature for a desired time, such as 10 minutes, and the presence of the recombinant lentiviral vector can be confirmed by the presence or absence of bands.

[0099] The cells containing the plasmid E6E7T obtained as above are streaked on an agar medium, and the formed colonies are picked and inoculated into a desired amount of a medium, for example, about 2 mL of LB medium containing antibiotics, and cultured with shaking at about 37° C. for about 8 hours with vigorous stirring. Next, a desired amount, for example, 100 μL of the culture solution of the cells cultured as above is transplanted into, for example, about 50 mL of LB medium containing a different antibiotic, for example, ampicillin, and cultured with shaking at about 37° C. for about 14 to 18 hours with vigorous stirring.

[0100] The amount of plasmid eluted with water is measured using a commercially available kit, such as MN NucleoBond Xtra Medium Extraction Kit (manufactured by Clontech). At the same time, analysis is performed using a desired gel, such as an approximately 1% agarose gel, to confirm that the inserted DNAs are different from each other. As markers for gel electrophoresis analysis, for example, supercoiled DNA ladder markers, λ-Hind III digested DNA, etc. can be used. Viral vectors can be produced as described above.

[0101] 2. Preparation of mammalian stem cells

[0102] 2.1 Preparation of porcine dental pulp stem cells

[0103] The jawbone (mandibular bone with mandibular teeth) of a pig 5 to 6 months after birth was obtained. Porcine dental pulp stem cells were obtained from the teeth and mandibular bone of the pig according to the following procedure.

[0104] First, the teeth and mandible of the pig are disinfected with an appropriate disinfectant, such as povidone-iodine. Then, the crown of the mandibular tooth is cut horizontally using, for example, a dental diamond bur, and then the pulp cavity roof is removed by cutting vertically. The dental pulp is collected from the crown and root using, for example, a dental surgical scraper.

[0105] The obtained dental pulp is minced using, for example, an ophthalmic puncture knife, suspended in a collagenase solution of a desired concentration, for example, 1 to 3 mg / mL, and placed in a thermostatic chamber at about 37° C. for a desired time, for example, 1 hour, to separate cells. The separated cells are pre-cultured in a desired culture medium, for example, DMEM containing 10% FBS and 1% antibiotic-antimyotic (Antibiotic-Antimyotic, manufactured by Invitrogen, Carlsbad, CA) at about 37° C. in a 5% CO 2 incubator to obtain cells for passage.

[0106] First, the cells are cultured while changing the medium 2 to 3 times a week until they reach near confluence, and the cells that have reached near confluence are detached using a detaching agent, such as a Hepes solution containing 0.05% trypsin, and collected under desired conditions, such as centrifugation at 1,500 rpm for about 3 minutes at room temperature. The obtained cells are transferred to fresh medium and subcultured under the same conditions as above using a desired amount, such as the entire amount.

[0107] 2.2 Preparation of human deciduous tooth pulp stem cells

[0108] Obtain deciduous teeth that have fallen out or been extracted from healthy children. After disinfecting such deciduous teeth using an appropriate disinfectant, such as a povidone-iodine solution, recover the pulp tissue in the same manner as when obtaining pig dental pulp. Digest the obtained pulp tissue in a solution of collagenase and dispase at the desired concentration (e.g., about 3 mg / mL of type I collagenase and about 4 mg / mL of dispase) at the desired temperature for the desired time, such as about 1 hour at about 37°C. Next, filter the solution using, for example, a 70 mm cell strainer (manufactured by Falcon) to filter out the cells.

[0109] The cells thus filtered out are resuspended in a desired amount, for example, 4 mL of the above-mentioned culture medium, and inoculated in a desired diameter, for example, a 6 cm diameter culture dish for adherent cell culture. A desired culture medium, for example, DMEM containing about 10% FCS, is added thereto, and cultured in an incubator adjusted to 5% CO2 and 37°C for a desired period of time, for example, about 2 weeks. The adherent cells (dental pulp stem cells) that have formed colonies are treated with a desired stripping agent, for example, about 0.2 mM EDTA containing about 0.05% trypsin at about 37°C for a desired period of time, for example, 5 minutes, and the cells stripped from the culture dish are recovered.

[0110] Next, the adherent cells recovered as above are seeded in, for example, a culture dish for adherent cell culture (a culture dish coated with collagen), and primary culture is performed in, for example, an incubator adjusted to 5% CO2 and 37°C to obtain primary cultured cells. When near confluence or confluence is reached by visual observation, the cells contained in the culture dish are recovered by treating in the same manner using the same stripping agent as described above.

[0111] Then, the above-mentioned culture medium is used to generate the desired concentration, for example, about 1×10 4 Cells / cm 2 The primary cultured cells were subcultured at a concentration of , and the cells subcultured 1 to 3 times were used in the experiment. As described above, human exfoliated deciduous tooth dental pulp stem cells (SHED) can be obtained.

[0112] If necessary, the porcine deciduous tooth dental pulp stem cells and human deciduous tooth dental pulp stem cells obtained as described above can be placed in vials and cryopreserved at -80 degrees.

[0113] 3. Preparation of Gene-Transferred Cells

[0114] 3.1 Gene transfer using lentiviral vectors

[0115] The porcine dental pulp stem cells and human dental pulp stem cells obtained as above are cultured in the same manner as above, and when the confluence reaches about 70% to 90%, mycoplasma detection is performed using a commercially available kit. As such a kit, for example, MycoAlert Mycoplasma Detection Kit (manufactured by Lonza, LT07-318) can be used. Next, the lentiviral vector prepared as above is infected with each of the above stem cells using a polybrene reagent, and drug selection is performed to select a stable expression strain of the target gene.

[0116] The culture supernatant of each stem cell cultured as above is removed, and after washing with, for example, PBS (pH 7.4), the cells are peeled off using a desired peeling agent and recovered separately. As such a peeling agent, for example, StemPro (registered trademark: manufactured by GIBCO) can be used for pig deciduous dental pulp stem cells, and trypsin-EDTA can be used for human deciduous dental pulp stem cells.

[0117] The obtained cells are counted according to a conventional method, for example, at a rate of about 1×10 cells / well in each well of a 6-well plate (manufactured by CORNING) that has been subjected to tissue culture treatment (TC treatment). 5 Cells / well were seeded and cultured in a CO2 incubator at about 37°C for about 24 hours to confirm that the cells were uniformly present throughout the well.

[0118] After that, the culture supernatant is removed, and a desired medium containing a desired concentration of polybrene, for example, a DMEM medium containing about 8 μg / mL of polybrene and about 10% FBS, is added to the porcine deciduous dental pulp stem cells in a desired amount, for example, 750 μL / well. Next, the lentiviral vector solution obtained above is added to each well, for example, at about 250 μL / well.

[0119] In the case of human deciduous dental pulp stem cells, the same DMEM containing polybrene and FBS as above is added in a desired amount, for example, about 1.2 mL / well, and then the above lentiviral vector solution is added to each well in a desired amount, for example, about 400 μL / well.

[0120] Each microplate to which the viral vector has been added as described above is centrifuged under desired conditions, such as about 1,000×g at about 32°C for about 30 minutes, to allow the virus to infect the cells. Thereafter, the cells are cultured under desired conditions, such as in a CO2 incubator at about 37°C for about 4 to about 6 hours, and then a desired amount of culture medium, such as about 1 mL / well, is added to each well, and the cells are further cultured under desired conditions, such as in a CO2 incubator at 37°C for about 24 hours to perform gene introduction.

[0121] 3.2 Drug selection

[0122] The culture supernatant is removed from each well of the culture plate of the stem cells (pig dental pulp stem cells and human dental pulp stem cells) cultured as above, and a selection medium containing a desired concentration of a selection agent, such as about 0.4 mg / mL or about 0.8 mg / mL of Geneticin (G418, manufactured by GIBCO) is added to each well in a desired amount, such as about 2 mL / well, and replaced with the selection medium. Thereafter, the cells are cultured for a desired period of time, such as about 3 to about 5 days, while the medium is replaced, to select the gene-transfected cells.

[0123] A portion of each of the above cultured cells is used for cloning, and the remaining portion is further cultured in a selection medium as pool cells. Cloning can be performed as follows.

[0124] The cells are diluted to a desired concentration, for example, about 1×10 3cells / 4mL or about 5×10 3 Cells / 4 mL are inoculated into each culture dish, and then cultured in a CO2 incubator at about 37°C for about 24 hours. The formed colonies are marked from the back side of the culture dish, and when the number of colonies reaches the desired number, for example, about 100, the culture supernatant is removed and washed with PBS. A cloning ring is placed in the culture dish, and the cells of the colonies peeled off using a peeling agent are added to each well of a 48-well plate to which a desired amount of culture medium, for example, about 1 mL, is added.

[0125] 3.3 Preparation of total RNA

[0126] In order to confirm gene expression, total RNA was prepared using, for example, NucleoSpin RNA II (a kit manufactured by MACHEREY-NAGEL). Various buffers, circular filters, etc. used below were used as accessories of the above kit, and from the aspect of obtaining highly pure total mRNA, it is preferred to operate according to the operating manual attached to the kit.

[0127] The desired number, for example, about 5×10 5 The cell mass prepared by lysing each cell to prepare a lysate is added to a purple ring filter and centrifuged at desired conditions, such as about 11,000 × g for about 1 minute. After centrifugation, the filter is discarded, and a desired amount of ethanol, such as about 350 μL of about 70% ethanol, is added to the collection tube, and pipetting is performed a desired number of times, such as about 5 times.

[0128] Next, a desired amount, for example, about 700 μL of the resulting solution is taken and loaded into a light blue annular column set in a collection tube, and centrifuged at desired conditions, for example, at about 11,000×g for about 30 seconds to allow RNA to bind. After centrifugation, the column is set in a new collection tube, a desired amount of reagent, for example, about 350 μL of MDB is added, and centrifuged at desired conditions, for example, at about 11,000×g for about 1 minute to desalt.

[0129] After centrifugation, gently mix the desired amount, for example, about 10 μL of rDNA enzyme (rDNase) and about 90 μL of DNA enzyme reaction buffer to prepare a DNA reaction mixture, add the desired amount, for example, about 95 μL, to the column, and incubate at room temperature for about 15 minutes under desired conditions to digest the DNA. Next, add the desired amount, for example, 200 μL of buffer RA2 to the column, and centrifuge at desired conditions, for example, at about 11,000 × g for about 30 seconds to wash. Next, place the column in a new collection tube, add the desired amount, for example, about 700 μL of buffer RA3 to the column, and further centrifuge at desired conditions, for example, at about 11,000 × g for about 30 seconds to wash.

[0130] Next, the solution flowing out into the collection tube is discarded, and a desired amount, for example, about 250 μL of buffer RA3 is added to the column again, and the column is further centrifuged at desired conditions, for example, about 11,000×g for about 2 minutes to air-dry the silica membrane of the column. The column is placed in a collection tube of a desired size, for example, about 1.5 mL, and a desired amount, for example, about 60 μL of RNase-free water is added to the column, and the column is centrifuged at desired conditions, for example, about 11,000×g for about 1 minute to obtain a total RNA sample.

[0131] 3.4 Reverse transcription reaction

[0132] The total RNA sample obtained as above is reverse transcribed, followed by real-time PCR to obtain a PCR product. For reverse transcription, commercially available products such as PrimeScript RT kit (Perfect Real Time, manufactured by Takara Bio) are used, and reverse transcription can be performed according to the manual attached to such a kit.

[0133] Real-time PCR can be performed using a commercially available kit such as SYBR Premix Ex Taq. In addition, as standard genes used here, for example, pig β-actin and human β-actin can be cited, and as primers, for example, primers shown in sequence numbers 7 to 12 in the sequence table can be prepared and used.

[0134] First, the desired amount, for example, about 350 μL of SYBR PremixEx Taq II (x2), about 28 μL of primer mixture and about 266 μL of sterile distilled water are mixed to prepare a premix for real-time PCR. Next, the desired amount, for example, about 23 μL of the above premix is ​​dispensed into tubes for real-time PCR, and about 2 μL of template cDNA is added to each tube. Next, real-time PCR is performed under the desired conditions, for example, about 95°C for about 30 seconds, about 40 cycles of about 95°C for about 5 seconds + about 60°C for about 30 seconds. Next, it is dissociated under the conditions of about 95°C for about 15 seconds, about 60°C for about 30 seconds, and about 95°C for about 15 seconds, and the melting curve is obtained. The same operation is performed for human dental pulp stem cells.

[0135] The expression level of each gene in cells into which the gene has been introduced using each viral vector can be determined from the calibration curve prepared as described above. By performing calibration using the expression level of the internal standard gene, a result with good accuracy can be obtained.

[0136] In each stem cell into which the gene is introduced, the expression of the introduced gene is confirmed. Through this confirmation, the gene introduction efficiency of each viral vector prepared as above can be obtained. Next, single cell cloning of cells is performed by the gene introduction cells obtained as above (hereinafter sometimes referred to as "introduced gene stable expression cell group" or "pool cell").

[0137] First, the above pool of cells is seeded in a desired number in a culture dish of desired size, for example, at about 1×10 3 The cells are inoculated in a 60 mm culture dish at a concentration of 100 cells / dish, and cultured in a CO2 incubator at a desired temperature for a desired time, for example, at 37°C for 24 hours. The culture medium is then replaced with the above-mentioned growth medium containing the selection agent, and the culture is continued to form colonies. For the formed colonies, each colony is peeled off using a cloning ring and a peeling agent, and is seeded in a desired microplate, for example, a 24-well plate.

[0138] After the inoculated cells are proliferated, they are inoculated in a culture dish or the like to proliferate and expand the culture, thereby obtaining a gene-introduced stable cell in a monoclonal form. Next, the obtained clone is cultured in the same manner as above to proliferate and obtain total mRNA. Using the obtained total RNA as a template, a desired kit, such as a PrimeScript RT kit, is used to perform a reverse transcription reaction to obtain a template cDNA.

[0139] Then, for example, real-time PCR is performed in the same manner as above using cDNA (reverse transcription product) as a template, the above-mentioned SYBR Premix Ex TaqII (Tli RNase H Plus), and primers (primers specific to the introduced gene and the internal standard gene, respectively, such as primers shown in sequence numbers 7 to 12 in the sequence listing).

[0140] The Ct value calculated from the second-order differential curve of the real-time PCR was plotted on the X-axis and the relative value of total RNA on the Y-axis to prepare a calibration curve for measuring the expression level of each gene. The relative values ​​of the expression levels of the introduced genes derived from pig dental pulp stem cells and human dental pulp stem cells were calculated when the expression level of the pool cells was set to 1, thereby determining whether all the introduced genes were expressed, and determining their expression levels to select the target clones.

[0141] As described above, it is possible to obtain an immortalized stem cell population into which a desired gene has been introduced, and clones of immortalized stem cells cloned from the cell population.

[0142] Ampicillin-resistant heterogeneous transformants (hereinafter referred to as "Amp+") are selected from the obtained transformants using, for example, ampicillin and purified, and restriction enzyme sites are analyzed to identify recombinants. Next, the recombinant adenovirus is digested using, for example, Pac I, and the resulting fragments are transfected into HEK293 cells to proliferate, which are collected and the titer of the virus is measured. The virus is purified according to conventional methods and infected with dental pulp stem cells as target cells.

[0143] According to the conventional method, the virus-infected HEK293 cell group was stained with FITC, and STRO-1 positive cells were detected using flow cytometry. Here, STRO-1 is considered to be one of the markers of mesenchymal stem cells with multidirectional differentiation potential in the bone marrow and an indicator of cell immortalization. Through the above process, immortalized stem cells derived from dental pulp can be obtained.

[0144] Next, the immortalized stem cells are cultured for 24 to 48 hours using the above-mentioned basal medium, for example, DMEM supplemented with 10% FBS, under the conditions of 5% CO2 and 37°C to obtain a culture supernatant. The culture supernatant can be recovered using, for example, a Komagome pipette.

[0145] In addition, the immortalized stem cells secrete growth factors such as insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), tissue metalloproteinases (TIMPs) and hepatocyte growth factor (HGF) into the culture supernatant. Here, "growth factor" is a general term for polypeptides that promote cell division, morphological changes or hypertrophy. Factors vary depending on the type of cells that produce the growth factor, and are roughly divided into epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), tumor growth factor (TGF), etc.

[0146] In addition, the receptors located on the cell membrane of each cell have tyrosine kinase activity. When the growth factor binds, the tyrosine residues of the protein are phosphorylated, causing the proliferation or differentiation of the cell. It is known that growth factors become several examples of mesoderm-inducing substances in individual occurrence. In addition, it is known that lymphokines that regulate the immune system become several examples of mesoderm-inducing substances in individual occurrence. Such growth factors can be quantitatively measured using the well-known ELISA method, microarray method, etc.

[0147] The above-mentioned IGF is a polypeptide with a highly similar sequence to insulin, and causes mitogenic reactions in cell culture in the same way as insulin. It is known that it also affects the growth of nerve cells. In addition, the above-mentioned VEGF is a group of glycoproteins related to angiogenesis (forming new blood vessels in a place without blood vessels during embryogenesis) and vasculogenesis (forming blood vessels by extending branches from existing blood vessels). HGF has multiple physiological activities not only for hepatocytes but also for various cells, such as promoting cell movement, resisting apoptosis (cell death), inducing morphogenesis, angiogenesis, and regeneration and protection of tissues / organs.

[0148] The above-mentioned various stem cells are cultured in DMEM supplemented with 15% FCS at 37°C for a predetermined period of time to obtain a culture supernatant containing the above-mentioned growth factors. The culture supernatant of the above-mentioned stem cells contains various proteins in addition to IGF, VEGF and HGF.

[0149] 15 mL of the obtained culture supernatant was added to an Amicon Ultracentrifugal Filter Units 10K (Millipore), centrifuged at 4,000 × g for about 60 minutes, and concentrated to about 200 μl. Next, an equal amount of sterilized PBS as the culture supernatant was added to the tube, and centrifuged again at 4,000 × g for about 60 minutes to replace the solvent with PBS. The obtained 200 μL solution was recovered in a microtube to prepare a concentrated stem cell culture supernatant.

[0150] Instead of the above-mentioned method using Amicon, ethanol precipitation can also be used for concentration. For example, 45 mL of 100% ethanol is added to 5 mL of culture supernatant, mixed, and left at -20°C for 60 minutes. Then, centrifuge at 15,000×g for 15 minutes at 4°C and remove the supernatant.

[0151] Next, add, for example, 10 mL of 90% ethanol and stir well, and centrifuge again at 4°C at 15,000×g for 5 minutes. Remove the supernatant, and the resulting mass can be dissolved in, for example, 500 μL of sterilized water. After dissolution, the entire amount is recovered in a micro test tube to prepare a concentrated stem cell culture supernatant. If the culture supernatant obtained as above is freeze-dried according to a conventional method, a particle powder of a composition for treating demyelinating diseases and peripheral neuropathy prepared at the time of use can be obtained.

[0152] For the reason of being able to restore demyelinating diseases and peripheral neuropathy or alleviate symptoms, it is preferred that the above-mentioned composition is a composition that can induce the regeneration of myelin or the proliferation of Schwann cells. Here, "Schwann cells" refer to cells that are present around the axons of peripheral nerve cells and cover the axons or axon terminals, and "myelin" refers to a structure formed by the above-mentioned Schwann cells wrapping around the axons of nerve cells, which assists the electrical signal transduction of nerve cells through the insulating effect. In addition, in addition to the above-mentioned insulating effect, it is also known that the cytokines secreted by Schwann cells are involved in the regeneration of nerve cells. Schwann cells can be obtained by primary culture of various experimental animals or by purchasing commercially available cell lines such as IMS32 or IFRS.

[0153] As the immortalized mesenchymal stem cells, dental pulp stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord and umbilical cord blood mesenchymal stem cells, etc. can be cited. Dental pulp stem cells are preferably used because of their high differentiation ability into nerves. In particular, when deciduous teeth are used, the obtained undifferentiated cells are more numerous and the regeneration effect of the above-mentioned myelin sheath or Schwann cell regeneration is high, so it is preferred.

[0154] In addition, when the composition obtained as above that can induce the regeneration of myelin or the proliferation of Schwann cells (hereinafter sometimes referred to as the "regenerative composition") contains at least a component that acts on the receptor c-MET of hepatocyte growth factor (HGF), HGF etc. can activate Schwann cells and promote the regeneration of peripheral nerves, it is preferred.

[0155] The pharmaceutical preparation for treating demyelinating diseases and peripheral neuropathy of the present invention contains the above-mentioned regeneration composition as an active ingredient, because the therapeutic effect is high and thus it is preferred. In addition, when the total weight of the preparation is set to 100%, the content of the above-mentioned regeneration composition is preferably 10 to 90% by weight. When the total weight of the preparation is set to 100%, the content of the above-mentioned regeneration composition is further preferably 25 to 75% by weight.

[0156] The above-mentioned pharmaceutical preparation can be appropriately used for demyelinating diseases caused by, for example, autoimmune reactions (i.e., immune reactions in which antibodies, T cells, and other immune cells induced by infection against pathogens also react to myelin sheaths and axons), or peripheral neuropathy caused as a side effect of the treatment of other diseases. As the above-mentioned demyelinating diseases, for example, Guillain-Barré syndrome, Fisher syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, MAG antibody-positive neuropathy, multifocal motor neuropathy, diabetic neuropathy, and postherpetic neuralgia can be cited. In addition, the above-mentioned pharmaceutical preparation can also be appropriately used for peripheral neuropathy caused as a side effect of the treatment of other diseases, such as peripheral neuropathy induced by chemotherapy for cancer.

[0157] The dosage form of the above-mentioned pharmaceutical preparation of the present invention is not particularly limited. According to the dosage form, suitable excipients, fillers, lubricants, pH regulators, etc. that are pharmacologically allowed can be added, or mixed with absorption enhancers, bases, etc. to make dosage forms such as powders, tablets, capsules, liquids, injections, and patches. In order to prepare such preparations, the above-mentioned regeneration composition can be made into freeze-dried powder. In addition, a sustained-release preparation can be made as required. The pharmaceutical preparation of the present invention can be manufactured as described above.

[0158] Example

[0159] The present invention is specifically described below by way of examples, but the present invention is not limited to the following examples.

[0160] (Example 1) Preparation of Viral Vectors for Immortalized SHED Production

[0161] 1. Preparation of adenovirus vector for transfection

[0162] (1) Reagents, etc.

[0163] (1-1) Plasmid extraction reagents

[0164] Kanamycin (Kan), ampicillin (Amp), LB liquid medium and LB agar medium, glycogen, agarose, sterile water, ammonium acetate, sodium acetate, sodium dodecyl sulfate and RNase A were used. 50 mg / mL of kanamycin (Kan) and ampicillin (Amp) were prepared and stored at -20°C in the form of stock solutions. Glycogen was prepared to 20 mg / mL. 10 mg / mL of RNase A was prepared and stored at -20°C. 10 M (saturated) ammonium acetate (NH4OAc) and 3 M sodium acetate (NaOAc; pH 5.2) were prepared.

[0165] (1-2) Restriction enzymes, etc.

[0166] E. coli competent cells (Supercharge EZ10 electroporation competent cells, product number 636756), SwaI (product number 1111A, Smi I is equivalent), Xho I (product number 1094A), T4 DNA ligase (product number 2011A), NucleoBond Xtra Midi (product number 740410.10 / .50 / .100), and NucleoSpin plasmid (product number 740588 10 / 50 / 250) were purchased from Takara Bio Co., Ltd. Pac I was purchased from New England Biolabs.

[0167] (1-3) Buffer solution, etc.

[0168] Prepare 1×TE buffer (10 mM Tris-HCl [pH 8.0] containing 1 mM EDTA), phenol:chloroform:isoamyl alcohol (25:24:1, hereinafter referred to as "PCI mixture") saturated with 100 mM Tris-HCl (pH 8.0). Use 100% and 70% ethanol. Prepare the following buffers 1 to 4 for purification of pAdeno-X plasmid DNA used in small-scale recombination.

[0169] Buffer 1: 25 mM Tris-HCl (pH 8.0) containing 10 mM EDTA and 50 mM glucose (stored at 4°C after autoclaving)

[0170] Buffer 2: 0.2 M NaOH containing 1% SDS (prepare immediately before use; seal and store at room temperature)

[0171] Buffer 3: 5M KOAc (sterilize at 4°C)

[0172] Buffer 4: 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA, 20 μg / mL RNase (add RNase immediately before use; store at -20°C)

[0173] (2) Reagents for adenovirus purification and β-gal detection

[0174] Human HEK293 cells (ATCC#CRL1573) transformed with human adenovirus type 5 were used. HEK293 cells were cultured in complete medium. The composition of the complete medium is DMEM (Double's modified Eagle's medium, basal medium) supplemented with 100 units / mL of penicillin G sodium, 100 μg / mL of streptomycin, 4 mM glutamine and 10% FBS. The penicillin G sodium solution was prepared at 10,000 units / mL, and the streptomycin sulfate solution was prepared at 10,000 μg / mL and stored in the form of a stock solution. 60 mm microplates, 100 mm microplates, 6-well plates, T75 and T175 flasks were used for culture.

[0175] Trypsin-EDTA (product number CC-5012) was purchased from Takara Bio Co., Ltd. Phosphate buffered saline (PBS, Ca-free) was prepared. 2+ and Mg 2+ ) and Dulbecco's phosphate buffered saline (DPBS, containing Ca 2+ and Mg 2 +). In addition, 0.33% neutral red staining solution and 0.4% trypan blue staining solution were used. β-gal luminescent detection kit (Luminescent β-gal Detection Kit) II (Product No. 631712, manufactured by Takara Bio Co., Ltd.) was used for β-gal detection.

[0176] (3) Preliminary test

[0177] (3-1) Construction of a recombinant adenovirus vector containing lacZ (pAdeno-X-lacZ)

[0178] After thawing, remove the DMSO from the HEK293 cells and resuspend them in 10 mL of the above complete medium. Transfer the entire amount to a culture plate with a diameter of 100 mm. After the HEK293 cells adhere to the wall, remove the culture medium, wash the cells once with sterile PBS, add 1 mL of trypsin-EDTA solution, and treat for about 2 minutes. Then, add 10 mL of complete medium to terminate the trypsin reaction and gently suspend. Count the viable cells and place 10 5 The cells were transferred to a culture plate with a diameter of 100 mm in which 10 mL of culture medium was added and spread evenly.

[0179] Using pShuttle2-lacZ (positive control vector included in Adeno-X expression system 1) and Adeno-X virus DNA included in the kit (digested with PI-Sce I and I-Ceu I), a recombinant adenovirus containing lacZ was constructed according to the protocol attached to the kit. The target cell SHED was infected, and the expression of β-galactosidase was detected to confirm the construction of the vector.

[0180] (3-2) Construction of recombinant pShuttle 2 plasmid

[0181] Before the construction of the recombinant pShuttle 2 vector (hereinafter referred to as "rpShuttle 2 vector"), DH5α Escherichia coli was transformed using the pShuttle 2 vector and pShuttle 2-lacZ vector included in the kit. Transformants were selected on LB agar plates containing 50 μg / mL kanamycin (hereinafter referred to as "LB / Kan"), and the cells obtained from a single colony were streaked on a new LB / Kan and incubated overnight at 37°C.

[0182] Next, hTERT, bmi-1, HPV-E6, and HPV-E7 were cloned into pShuttle 2 according to the following process. The pShuttle 2 vector was cut using restriction enzymes suitable for these genes. Next, referring to the pShuttle 2 vector information package (PT3416-5) attached to the above kit, a multicloning site matching the inserted DNA was determined. The above plasmid treated with restriction enzymes was treated with alkaline phosphatase and purified.

[0183] Prepare the target DNA fragment and purify it according to the conventional method. Connect the vector digested with the restriction enzyme and the gene fragment, and transform DH5α cells (competent cells) with the ligation product. Take a part of the competent cells and transform them with the control vector pShuttle2-lacZ vector included in the kit as a positive control.

[0184] The mixture containing the transformed E. coli was inoculated on LB / Kan agar plates to select kanamycin-resistant (Kanr) transformants (colonies). 5 to 10 Kan-resistant clones were selected and inoculated into a small amount of liquid culture medium for amplification. After confirming that these clones have the rpShuttle 2 vector, incubate overnight. Afterwards, the constructed plasmid DNA was purified using a commercially available silica adsorption column according to conventional methods.

[0185] The plasmid DNA was treated with restriction enzymes and subjected to 1% agarose gel electrophoresis to identify the target recombinant plasmid. The direction and insertion site of the inserted fragment were confirmed by sequencing to identify positive clones. The recombinant pShuttle2 plasmid DNA (hereinafter referred to as "rpShuttle2 plasmid DNA") was directly transfected into target cells and protein immunoblotting was performed to pre-detect the expression of the target protein.

[0186] (3-3) PI-Sce I / I-Ceu I double digestion of rpShuttle 2 plasmid DNA

[0187] The expression cassette of the introduced gene was cut out from the rpShuttle2 plasmid DNA prepared as above using PI-Sce I and I-Ceu I. The cut expression cassette was inserted into the Adeno-X viral DNA according to the in vitro ligation method described in the operating procedures attached to the kit. 30 μl of PI-Sce I / I-Ceu I double digestion solution of rpShuttle 2 plasmid DNA was prepared, and the reagents described in Table 1 below were placed in a 1.5 mL sterilized microcentrifuge tube and mixed.

[0188] [Table 1]

[0189]

[0190] Next, after thorough mixing, the mixture was placed in a microcentrifuge tube and gently centrifuged, followed by incubation for 3 hours at 37° C. The double-digested reaction solution (5 μL) was electrophoresed together with a 1 kb ladder marker (DNA size marker) in a 1% agarose / EtBr gel.

[0191] (3-4) Phenol:chloroform:isoamyl alcohol extraction

[0192] Add 70 μL of 1×TE buffer (pH 8.0) and 100 μL of PCI mixed solution to the remaining double digestion solution (25 μL) in a centrifuge tube, and stir thoroughly with a vortex mixer. Next, centrifuge at 14,000 rpm for 5 minutes at 4°C using a microcentrifuge, and transfer the aqueous layer to a clean 1.5 mL microcentrifuge tube. Add 400 μL of 95% ethanol, 25 μL of 10 M ammonium acetate, and 1 μL of glycogen (20 mg / mL), and stir thoroughly with a vortex mixer.

[0193] Next, the mixture was centrifuged at 14,000 rpm for 5 minutes at 4°C, and the supernatant was removed by suction to obtain a pellet. 300 μL of 70% ethanol was added to the pellet, and the pellet was centrifuged at 14,000 rpm for 2 minutes at room temperature. The supernatant was carefully removed by suction, and the pellet was air-dried at room temperature for about 15 minutes.

[0194] After the pellet was dried, it was dissolved in 10 μL of 1× TE sterile buffer (pH 8.0) and stored at -20°C before use.

[0195] (4) Construction of recombinant Adeno-X plasmid DNA

[0196] (4-1) Subcloning of the expression cassette into the Adeno-X viral genome

[0197] The reagents shown in Table 2 below were added sequentially to a 1.5 mL sterilized microcentrifuge tube, mixed gently, centrifuged gently, and incubated at 16°C overnight.

[0198] [Table 2]

[0199] Reagents, etc. Liquid volume (μL) pShuttle 2 plasmid DNA after PI-Sce I / I-Ceu I digestion 2.0 pShuttle 2-lac Z plasmid DNA after PI-Sce I / I-Ceu I digestion - Sterilized water 3.0 10× DNA Ligation Buffer 1.0 Adeno-X virus DNA (250ng / μL) 3.0 DNA ligase (1 unit / μL) 1.0 total 10.0

[0200] 90 μL of 1×TE buffer (pH 8.0) and 100 μL of PCI mixed solution were added to each sample, and the mixture was gently stirred using a vortex mixer. The mixture was centrifuged at 14,000 rpm for 5 minutes at 4°C, and the aqueous layer was transferred to a clean 1.5 mL microcentrifuge tube, to which 400 μL of 95% ethanol, 25 μL of 10 M ammonium acetate, and 1 μL of glycogen (20 mg / mL) were added, and the mixture was gently stirred using a vortex mixer.

[0201] The pellet was centrifuged at 14,000 rpm for 5 minutes at 4°C, and the supernatant was removed by suction to obtain a pellet. The subsequent ethanol precipitation operation was performed in the same manner as in the above (3-4). After the pellet was dried, it was dissolved in 15 μL of sterile deionized water.

[0202] (4-2) Swa I digestion of recombinant Adeno-X plasmid DNA

[0203] The digestion solutions shown in the following Table 3 were prepared, added to each sample in a centrifuge tube, and incubated at 25°C for 2 hours.

[0204] [Table 3]

[0205] Reagents, etc. Liquid volume (μL) Connect products 15.0 10× Swa I digestion buffer 2.0 10×BSA 2.0 Swa I (10 units / μL) 1.0 total 20.0

[0206] 80 μL of 1×TE buffer (pH 8.0) and 100 μL of PCI mixed solution were added to each sample and gently stirred using a vortex mixer. Centrifuge at 14,000 rpm for 5 minutes at 4°C using a microcentrifuge tube. The subsequent ethanol precipitation operation was performed in the same manner as in (3-4) above, and the pellet solution was stored at -20°C before use.

[0207] (4-3) Confirmation of E. coli transformation using recombinant Adeno-X plasmid DNA

[0208] Using Supercharge EZ10 electroporation competent cells (Product No. 636756), the Swa I digestion product obtained in (4-2) was used to transform the competent cells for electroporation (E. coli). The transformation mixture was inoculated on an agar plate (hereinafter referred to as "LB / Amp agar plate") in which ampicillin (final concentration 100 μg / mL) was added to LB medium and incubated overnight at 37°C. About 10 transformants with ampicillin resistance (Ampr) were obtained. 6 The obtained colonies were detected using the Adeno-X system PCR screening primer set attached to the product.

[0209] Bacteria from a single colony were inoculated into 5 mL of fresh LB / Amp liquid medium and cultured overnight. The next day, Adeno-X plasmid DNA was purified according to the small-scale method described below.

[0210] (5) Small-scale preparation of recombinant Adeno-X plasmid DNA

[0211] Centrifuge 5 mL of the culture solution in the logarithmic growth phase at 14,000 rpm for 30 seconds and remove the supernatant. Centrifuge the pellet again at 10,000 rpm for 1 minute and remove the supernatant using a micropipette. Add 150 μL of the above buffer 1 and gently pipette to resuspend. Add 150 μL of buffer 2 to the cell suspension, gently invert to mix, and place on ice for 5 minutes. Add 150 μL of buffer 3 to the cooled cell suspension, invert again to mix, and place on ice for 5 minutes.

[0212] The cell suspension was centrifuged at 14,000 rpm for 5 minutes at 4°C, and the clear supernatant was transferred to a clean 1.5 mL centrifuge tube. 450 μL of PCI mixture was added to the supernatant, and the mixture was mixed by inversion. The mixture was then centrifuged at 14,000 rpm for 5 minutes at 4°C, and the aqueous layer was transferred to a clean 1.5 mL microcentrifuge tube.

[0213] The subsequent ethanol precipitation operation was performed in the same manner as in the above (4-1), and the pellet solution was stored at -20°C until use. The target rDNA was identified by restriction enzyme analysis and PCR as described below.

[0214] (6) Restriction enzyme site analysis of the obtained rAdeno-X plasmid DNA

[0215] Analyze using PI-Sce I and I-Ceu I. Place the reagents shown in Table 4 below into a 1.5 mL sterilized microcentrifuge tube, add 30 μL of PI-Sce I / I-Ceu I double digestion reaction solution, stir thoroughly, rotate gently, and collect the contents. Incubate at 37°C for 3 hours and perform restriction enzyme treatment. Perform electrophoresis on a 1% agarose / EtBr (ethidium bromide) gel.

[0216] [Table 4]

[0217] Reagents, etc. Liquid volume (μL) Sterilized water 19.5 10× double digestion solution 3.0 rpAdeno-X DNA(500ng / μl)(500ng / μL) 2.0 pShuttle 2-lac Z plasmid (500ng / μL) - PI-Sce I (1 unit / μL) 2.0 I-Ceu I (5 units / μL) 0.5 10×BSA 3.0 total 30.0

[0218] (7) Production of recombinant adenovirus

[0219] (7-1) Preparation of rAdeno-X plasmid DNA for HEK293 cell transfection

[0220] The reagents and the like shown in Table 5 below were placed in a 1.5 mL sterilized centrifuge tube, mixed, and gently centrifuged using a microcentrifuge, and then incubated at 37°C for 2 hours to treat the rAdeno-X plasmid DNA with the Pac I restriction enzyme.

[0221] [Table 5]

[0222] Reagents, etc. Liquid volume (μL) Sterilized water 20 pAdeno-X plasmid DNA (500 ng / μL) 10 10× Pac I digestion buffer 4 10×BSA 4 Pac I (10 units / μL) 2 total 40

[0223] 60 μL of 1×TE buffer (pH 8.0) and 100 μL of PCI mixed solution were added, gently stirred with a vortex mixer, and centrifuged at 14,000 rpm for 5 minutes at 4° C. The aqueous layer was carefully transferred to a clean 1.5 mL sterilized centrifuge tube.

[0224] The subsequent ethanol precipitation operation was carried out in the same manner as in the above (3-4), and the pellet solution was stored at -20°C until use.

[0225] (7-2) Transfection of Pac I-digested Adeno-X plasmid DNA into HEK293 cells

[0226] 24 hours before the above plasmid DNA transfection, the number of cells per 60 mm diameter culture plate was 1×10 6 ~2×10 6 (About 100 cells / mm 2 HEK293 cells were inoculated in a 37° C., 5% CO 2 atmosphere.

[0227] 10 μL of Adeno-X plasmid DNA digested with Pac I was transfected into each culture plate, and Adeno-X DNA was introduced into HEK293 cells according to the standard transfection method (CalPhos Mammalian Transfection Kit, Product No. 631312, manufactured by Takara Bio Co., Ltd.). Confirm whether CPE (cell degeneration effect) has occurred since the day after transfection. After 1 week, the cells attached to the bottom and sides of the culture plate were gently stirred to free them. The resulting cell suspension was transferred to a 15 mL sterilized conical centrifuge tube and centrifuged at 1,500 × g for 5 minutes at room temperature.

[0228] The obtained precipitate was suspended in 500 μL of sterile PBS. The freeze-thaw operation of freezing in dry ice / ethanol and thawing in a thermostatic bath at 37°C was repeated three times to fully thaw the cells and obtain a lysate. Then, the suspended matter was removed by gentle centrifugation, and the supernatant was transferred to another sterilized tube for immediate use. The portion not used immediately was stored at -20°C. 250 μL of the above lysate was added to the cultured cells in a 60 mm culture plate and the culture was continued. It should be noted that the anti-Hexon antibody contained in the Adeno-X Rapid Titer Kit (Product No. 631028, manufactured by Takara Bio Co., Ltd.) was used to determine the titer of the adenovirus according to the operating instructions of the kit (PT3651-1).

[0229] (7-3) Virus Amplification for High-Titer Virus Preparation

[0230] About 24 hours before the start of the titer measurement, HEK293 cells were seeded into a T75 flask and cultured overnight at 37°C in the presence of 5% CO 2 to confirm that the cells had reached 50% to 70% confluence.

[0231] The next day, the medium was replaced with a new one containing virus, and infection was performed at an MOI of 10. After culturing at 37°C and 5% CO2 for 90 minutes, the flask was removed and 10 mL of the medium was added.

[0232] Cultivate at 37°C and 5% CO2 for 3-4 days to confirm CPE. When 50% of the cells are detached, prepare a free cell suspension in the same manner as above and transfer to a 15 mL sterilized conical centrifuge tube. Perform the same freeze-thaw operation as above to thaw the cells. Use the Adeno-X Rapid Titer Test Kit (Product No. 631028) to obtain 10 7 Western blotting was performed to confirm whether the packaged adenoviral genome contained copies of the target gene-specific transcription unit in a functional form.

[0233] (8) Adenovirus infection of target cells

[0234] (8-1) Infection of target cells

[0235] 24 hours before infection, 1×10 6 SHEDs were inoculated into 6-well plates. The culture medium was removed the next day after inoculation, and 1.0 mL of culture medium containing virus was added to the center of each microplate. The solution was spread evenly over the entire monolayer formed by SHEDs.

[0236] The virus was infected in SHED by culturing at 37°C and 5% CO 2 for 4 hours. Fresh medium was then added and the cells were further cultured at 37°C and 5% CO 2 . The expression of the introduced gene was analyzed over time 24 to 48 hours after infection.

[0237] (8-2) Analysis of β-galactosidase expression in infected cells

[0238] The expression of β-galactosidase in adherent cells infected with Adeno-X-lacZ was detected using β-gal Luminescent Detection Kit II (Product No. 631712, manufactured by Clontech).

[0239] 2. Preparation of Retroviral Vectors

[0240] (1) Reagents, etc.

[0241] G3T-hi cells (manufactured by Takara Bio, product number 6163), TransIT-293 (transfection reagent, manufactured by Takara Bio, product number MIR2704), retrovirus packaging kit Ampho (manufactured by Takara Bio, product number 6161), retrovirus titer detection kit (for real-time PCR) (manufactured by Takara Bio, product number 6166), one-step SYBR PrimeScript RT-PCR kit (Perfect RealTime, manufactured by Takara Bio, product number RR066A) were used. In addition, a real-time PCR device Thermal Cycler Dice Real System (manufactured by Takara Bio, model TP800) was used.

[0242] (2) Plasmid preparation

[0243] In order to prepare a retroviral plasmid for retroviral vector preparation, pDON-5Neo (manufactured by Takara Bio, product number 3657) was used. According to conventional methods, a Kozak sequence (gccacc) was added upstream of the start codon of TERT (human or porcine, hereinafter sometimes referred to as "hTERT" or "pTERT"), E6 and E7 of human papillomavirus, and hBmi-1 as an immortalization gene.

[0244] The above five immortalization genes with Kozak sequences attached were cloned into the Pmac I-Hpa I site of the above pDON-5Neo DNA, and 10 mL each of recombinant retroviral vectors (amphitropic envelope) carrying pDON-5Neo hTERT vector (SYN5587-1-4), pDON-5Neo HPV16E6 vector (SYN5587-2-10), pDON-5Neo HPV16 E7 vector (SYN5587-3-7), pDON-5Neo porcine TERT vector (SYN5587-4-9), and pDON-5Neo hBmi1 vector (SYN5587-5-1) were prepared.

[0245] The plasmid DNA obtained as above was confirmed by single-strand analysis of the inserted gene sequence. The plasmid DNA was used to transform Escherichia coli to obtain a transformant. The obtained transformant was cultured, and then the transfection-grade plasmid DNA (about 50 μg) was purified according to conventional methods to obtain a plasmid DNA solution in the form of a DNA solution dissolved in sterile water.

[0246] (3) Production of recombinant retroviral vectors

[0247] Five tissue culture dishes (100 mm in diameter) were used, and 6 × 10 6 G3T-hi cells were cultured at 37°C for approximately 24 hours, and three plasmids for retrovirus production (any vector selected from the group consisting of pDON-5Neo hTERT vector, pDON-5Neo HPV16E6 vector, pDON-5Neo HPV16E7 vector, pDON-5Neo porcine TERT vector, and pDON-5Neo hBmi1 vector, and pGP and pE-Ampho attached to the retrovirus packaging kit Ampho) were co-introduced into each culture dish using a transfection reagent, and the cells were further cultured at 37°C for 48 hours.

[0248] After the culture was completed, the culture supernatant containing the retroviral vector was recovered and sterilized by filtration using a 0.45 μm filter (Millipore), and 1 mL of the filtrate was dispensed into sterile tubes.

[0249] (4) Calculation of the titer of recombinant retroviral vector

[0250] The titer of the recombinant retroviral vector was quantified using the Retroviral Titer Detection Kit (for Real-Time PCR) and the One-Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) according to the instructions attached to the kit. The template treatment and real-time PCR reaction conditions for the titer determination of the retroviral vector are shown in Tables 6 and 7 below.

[0251] The composition of the reaction solution used in the template treatment (template treatment with DNase I) in the titer measurement of the retroviral vector is shown in Table 6 below. The reaction conditions are shown in Table 7 below. That is, the solution shown in Table 6 below was incubated at 37°C for 30 minutes, then heated to 70°C and maintained for 10 minutes, and then rapidly cooled to 4°C.

[0252] [Table 6]

[0253]

[0254] Real-time PCR was performed under the following reaction conditions using the solutions shown in Table 7: (s1) 42°C for 5 minutes, (s2) 95°C for 10 seconds, and then (s3) 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds to prepare a melting curve.

[0255] [Table 7]

[0256] Reagents Dosage / Reaction 2x One-Step SYBR RT-PCR Buffer III 12.5μL TaKaRa ExTaq HS (5U / μL) 0.5μL PrimeScript RT Enzyme Mix II 0.5μL Forward titer primer FRT-1 (10 pmol / μL) 0.5μL Reverse titer primer FRT-1 (10 pmol / μL) 0.5μL <![CDATA[dH2O free of RNase]]> 8.5μL template 2μL total 25.0μL

[0257] The copy number of the RNA control template included in the kit is taken as the X-axis and the second-order differential curve (second-order derivative (2 nd The Ct values ​​calculated by the real-time PCR were plotted on the Y axis to create a calibration curve. The following Table 8 shows the Ct values ​​of the calibration curve for real-time PCR. In the following table, for example, "1.00E+08" means "1.00×10 8 ". The same applies below.

[0258] [Table 8]

[0259]

[0260] The Ct value was calculated by the second derivative maximum (SDM) method, and the slope was -3.54, the intercept was 43.88, the amplification efficiency was 91.6, and the determination coefficient was 0.999. The titer of the test sample was calculated from the calibration curve, and the results are shown in Table 9.

[0261] [Table 9]

[0262]

[0263] In the above table, ED indicates that the dilution buffer EAST diluent (for real-time PCR) was used. In addition, the titer (copy number / mL) was calculated as the copy number (copy number / μL) × 2 (dilution factor during DNase I treatment) × dilution factor during real-time PCR reaction (100 or 500) × 1,000 (converted from μL to mL).

[0264] According to the results of real-time PCR, pDON-5Neo hTERT showed a 1.46×10 10 The titer of pDON-5Neo HPV16E6 was 1.15×10 10 The titer of copies / mL for pDON-5Neo HPV16E7 was 4.05×10 10 The titer of pDON-5Neo porcine TERT was 1.27×10 10 The titer of copies / mL of pDON-5NeohBmi1 was 2.83×10 10 Titer in copies / mL.

[0265] (Example 2) Preparation of Immortalized SHED and Its Culture Supernatant

[0266] The deciduous teeth obtained from a 10-year-old healthy boy were used. After the deciduous teeth were disinfected with povidone-iodine solution, the crowns were cut horizontally using a dental diamond bur, and the dental pulp tissue was recovered using a dental reamer. The obtained pulp tissue was digested in a solution of 3 mg / mL type I collagenase and 4 mg / mL dispase at 37°C for 1 hour. Next, the solution was filtered using a 70 mm cell strainer (manufactured by Falcon).

[0267] The filtered cells were resuspended in 4 mL of the above culture medium and inoculated into a 60 mm diameter culture dish for adherent cell culture. DMEM (Double's modified Eagle's medium) containing 10% FCS was added to the culture dish and cultured in an incubator adjusted to 5% CO2 and 37°C for about 2 weeks. The adherent cells (dental pulp stem cells) that formed colonies were treated in 0.05% trypsin·0.2 mM EDTA at 37°C for 5 minutes, and the cells peeled off from the culture dish were recovered.

[0268] Next, the adherent cells selected as described above were inoculated into a culture dish for adherent cell culture (a culture dish coated with collagen), and primary culture was performed in an incubator adjusted to 5% CO2 and 37°C to prepare primary culture cells. When the cells reached near confluence (a state where the cells occupied about 70% of the surface of the culture container) or confluence as observed by the naked eye, the cells were treated with 0.05% trypsin-0.2mM EDTA at 37°C for 5 minutes, and the cells were detached from the culture container and recovered. The cells thus obtained were inoculated again into a culture dish containing the above-mentioned culture medium, and subcultured several times to proliferate to about 1×10 7 The obtained cells were stored in liquid nitrogen.

[0269] Then, the above culture medium was used to prepare about 1×10 4 Cells / cm 2 The primary cultured cells were subcultured at a concentration of 1.5 ng / mL. The cells after 1 to 3 subcultures were used in the experiment. Human exfoliated deciduous tooth dental pulp stem cells (SHED) were obtained as described above. About 1×10 SHED were sorted from each sample using FACSTARPLUS (manufactured by Becton-Dickinson) as follows. 6 STRO-1 positive cells.

[0270] According to the manufacturer's instructions of the Bromodeoxyuridine BrdU staining kit (manufactured by Invitrogen), BrdU uptake was performed for 12 hours to evaluate the proliferation rate of SHED (n=3 in each group). The experiment was repeated 5 times. One-way ANOVA followed by Tukey-Kramer test was performed to evaluate statistically significant differences.

[0271] To detect STRO-1 by immunofluorescence, SHED cells were fixed with 3% paraformaldehyde, rinsed twice with PBS, and treated with 100 mM glycine for 20 minutes. The cells were then permeabilized with 0.2% Triton-X (Sigma-Aldrich) for 30 minutes and incubated in a mixture of 5% donkey serum and 0.5% bovine serum albumin for 20 minutes.

[0272] Next, the cells were incubated with mouse anti-human STRO-1 antibody (1:100, manufactured by R&D) as a primary antibody for 1 hour, and with goat anti-mouse immunoglobulin M-FITC antibody (1:500, manufactured by SouthernBiotech) as a secondary antibody for 30 minutes, and covered with Vectashield DAPI (Vector Laboratories Inc.). α-MEM supplemented with 15% FBS was then added to a 6-well plate, and the sorted cells were inoculated for cloning. About 300 colonies of the proliferated cells were combined for the experiment.

[0273] (2) Gene introduction

[0274] (2-1) Gene transfer based on adenovirus vector

[0275] As described above, four genes, namely bmi-1, E6, E7 and hTERT, were inserted into adenoviral vectors to prepare viral vectors expressing these gene products. As a control, a control vector without these genes was prepared.

[0276] SHED was seeded in a collagen-coated culture dish with a diameter of 100 mm at 1 × 10 6 , add DMEM supplemented with 10% FBS, and culture until nearly confluent. Aspirate the culture medium, add 500 μL of virus solution diluted with the above culture medium (MOI=10), and culture at 37°C in a 5% CO2 incubator for 1 hour to infect the above virus vector. After 48 hours of infection, transfer the infected cells to the above culture medium supplemented with puromycin (1 pg / mL), culture for 10 days for selection, and merge 500 to 600 resistant clones. Every 3 to 4 days, about 0.5×10 5 SHEDs were inoculated into a culture dish with a diameter of 100 mm and passaged. The SHED into which the gene was introduced was designated SHED-T, and the SHED into which the gene was not introduced was designated SHED-C.

[0277] (2-2) Gene transfer based on retroviral vectors

[0278] The retroviral vector obtained as above was used to infect human dental pulp-derived cells (KA_01_W14_P5D3) or human dental pulp-derived cells (KA_02_W4-6_P4D3) to obtain drug-resistant pool cells (hereinafter referred to as "pool cells"). The obtained pool cells were subjected to real-time PCR analysis to confirm the number of inserted genes. Real-time PCR used the primers listed in Table 10 below (sequence numbers 13 to 16, all manufactured by Takara Bio).

[0279] [Table 10]

[0280]

[0281] The above KA_01_W14_P5D3 was cultured in a Corning adherent cell culture dish (6 cm or 10 cm diameter cell culture dish, surface treated with TC) at 37°C in a 5% CO2 incubator. The passage number when these cells were revived and inoculated was indicated as [0] (hereinafter indicated as "+P0"), the 1st passage was indicated as "+1", and the 2nd passage was indicated as "+2".

[0282] When the cells in the above-mentioned culture dish reach about 80% confluence, the culture supernatant is removed, and the cells are washed 2 to 3 times with PBS. Then, a 0.25% trypsin-EDTA solution (1x) (containing phenol red) (manufactured by Thermo Fisher Scientific, 25200-056) is added as a stripping agent, and incubated at 37°C in a 5% CO2 incubator for 3 minutes to peel the adherent cells from the bottom of the culture dish.

[0283] An appropriate amount of culture medium was added thereto and pipetted to prepare a cell suspension of a single cell. The cell suspension was diluted to an appropriate amount and passaged to a new culture dish. Thereafter, the same method was used to continue culturing, and it was confirmed that there was no abnormality in the proliferation and morphology of the cells. In addition, for the culture supernatant collected during passage, MycoAlert Mycoplasma Detection Kit (manufactured by Loza, product number LT07-318) was used to confirm that there was no mycoplasma contamination.

[0284] (3) Study on drug resistance concentration

[0285] In order to select gene-transfected cells using drugs, the G418 resistance concentration of target cells was studied. A 6-well plate from Corning (6-well TC-treated transparent multiwell plate, product number 3516) was used and 2.0×10 4Cells in logarithmic growth phase. Cultured in a 5% CO2 incubator at 37°C for about 24 hours, after which cells were confirmed to be attached to the bottom surface of the wells. The culture medium in each well was replaced with a culture medium supplemented with different concentrations of G418. The cell status was photographed 4 days, 8 days, and 10 days after the start of drug selection to study drug resistance concentrations.

[0286] It was confirmed that cells lost their proliferation activity in wells with a drug concentration of 250 μL / mL or more 4 days after the start of drug selection, and most cells died in wells with a drug concentration of 500 μL / mL or more 11 days after the start of drug selection. Based on the above, the G418 selection drug concentration for target cells was determined to be 500 μL / mL, which showed a sufficient selection effect.

[0287] The retroviral vector solution was used to infect the target cells by the polybrene method, and the immortalized gene stable expression strains were selected using medium B. 1.40×10 5 Cells in logarithmic growth phase were cultured at 37°C in a 5% CO2 incubator for about 24 hours. After that, polybrene (hexadimethrine bromide, manufactured by Sigma-Aldrich, product number H9268) was added to the recombinant retroviral vector solution diluted to 4 or 10 times with medium A at a final concentration of 8 μg / mL, and added equally to each well to infect the cells with the virus. About 4 hours after the virus infection, 1.0 mL of medium A was added to each well. The culture plate was incubated at 37°C in a 5% CO2 incubator, and medium B was replaced after 24 hours to start drug selection.

[0288] Ten days after the start of drug selection, cells that had become drug-resistant and proliferated were collected, and 1 mL of CELLBANKER 1Plus (manufactured by Zenyaku Industries, product number CB021) was added to prepare a 1×10 6 Cell suspension of 10 × 10 cells / mL was stored at -80°C as frozen cell stock solution. 5 Cell pellets of 100 cells / vial were used for real-time PCR analysis and stored at -80°C.

[0289] (4) Preparation of culture supernatant of immortalized stem cells

[0290] 2×10 immortalized dental pulp stem cells prepared above were 5 10 mL of DMEM supplemented with 10% FBS was added to a culture dish (55 cm 2 ) and cultured at 37°C in a 5% CO 2 incubator until reaching 70% confluence (4 to 6 days).

[0291] Confirm that 70% confluence has been achieved using a microscope, then use an aspirator to remove the culture supernatant in the culture flask, and wash twice with DMEM without serum. Then, add 10mL of DMEM without serum (hereinafter sometimes referred to as "normal DMEM") to the culture flask. The culture supernatant recovered immediately after addition is used as a control. After addition, culture at 37°C in a 5% CO2 incubator for 72 hours. The culture supernatant is collected in a 15mL sterile centrifuge tube, centrifuged at 440×g for 3 minutes, and the centrifugal supernatant is passed through a 0.22mm PVDF filter (manufactured by Millipore) to obtain the culture supernatant of immortalized SHED (hereinafter sometimes referred to as "SHED-CM").

[0292] (Example 3) Analysis of factors contained in culture supernatant

[0293] (1) Preparation of culture supernatant of immortalized stem cells

[0294] 2×10 immortalized stem cells prepared in Examples 1 and 2 were 5 The cells / mL were added to a culture dish (55 cm) containing 10 mL of Dulbecco's Modified Eagle Medium (hereinafter sometimes referred to as "DMEM") supplemented with 10% fetal bovine serum (hereinafter sometimes referred to as "FBS"). 2 ) and cultured at 37°C in a 5% CO 2 incubator until reaching 70% confluence (4 to 6 days).

[0295] After confirming that 70% confluence has been achieved using a microscope, the culture supernatant in the culture flask is removed using an aspirator and washed twice with DMDM ​​without serum added. Next, 10 mL of DMEM without serum added (hereinafter sometimes referred to as "normal DMEM") is added to the culture flask. The culture supernatant recovered immediately after addition is used as a control. After addition, culture at 37°C in a 5% CO2 incubator for 72 hours. The culture supernatant is collected in a 15 mL sterile centrifuge tube and centrifuged at 1,500 rpm (about 440 × g) for 3 minutes. The centrifugal supernatant is passed through a 0.22 mm PVDF filter (manufactured by Millipore) to obtain the culture supernatant of SHED-CM.

[0296] (2) Quantitative expression analysis

[0297] The cytokines expressed in the SHED-CM obtained above were quantitatively analyzed by the following procedure using Quantibody (registered trademark) Human Cytokine Antibody Array 1000 (RayBiotech Inc., product number QAH-CAA-1000) according to the attached instruction manual.

[0298] First, the SHED-CM was contacted with the antibody fixed on the array for 1 to 2 hours, and after washing, a mixture of biotinylated antibodies was added to each well and incubated for 1 to 2 hours. Then, labeled streptavidin was added and incubated for 2 hours, and the labeled signal was detected, and data analysis was performed using GenePix (registered trademark) 4400A (Molecular Devices, LLC). As a result, 80 cytokines were detected, which are shown in Table 11 below.

[0299] [Table 11]

[0300]

[0301] Insulin-like growth factor binding proteins IGFBP-4, IGFBP-6 and IGFBP-2, and tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 were found to be present in relatively high amounts. Insulin was excluded because it was not properly measured this time.

[0302] (3) Qualitative expression analysis

[0303] Regarding which cytokines are expressed in the SHED-CM obtained above, qualitative expression analysis was performed by the following procedure using RayBio (registered trademark) Human Cytokine Antibody Array G-Series 4000 (RayBiotech Inc., product number AAH-CYT-G4000-4) according to the attached instruction manual.

[0304] First, the SHED-CM or standard protein mixture was contacted with the antibody array fixed on the slide for 1 to 2 hours, and then incubated with the biotinylated antibody mixture for 1 to 2 hours. Then, it was incubated with an equal amount of Cy3 labeled streptavidin for 1 hour, and scanned, data extracted and analyzed using GenePix (registered trademark) 4400A (Molecular Devices, LLC).

[0305] This qualitative analysis detected 264 cytokines (Tables 12 to 16 below), of which 77 cytokines had relative expression intensities of 1.5 or more to the control.

[0306] [Table 12]

[0307]

[0308] [Table 13]

[0309]

[0310] [Table 14]

[0311]

[0312] [Table 15]

[0313]

[0314] [Table 16]

[0315]

[0316] According to the above results, the relative expression intensity of NGF, IL family (IL-6, IL-8, IL6R, IL-17C, IL-17R, etc.) is less than 10.

[0317] (Example 4) Verification of the effect of SHED-CM on autoimmune neuritis

[0318] The therapeutic effect of the SHED-CM prepared above was verified using experimental autoimmune neuritis (EAN) mice, which are disease model mice of Guillain-Barré syndrome. The model mice were 6-week-old C57BL / 6 male mice (purchased from Sankyo Labo Service Co., Ltd.), and were acclimated and raised for 1 week at room temperature with free access to water and feed in a 12-hour light-dark cycle, and divided into a negative control group and a treatment group (n=5).

[0319] After acclimation and breeding, a part of P0, one of the main constituent proteins of myelin sheath (P0 peptide (Biologica)) was dissolved in PBS to prepare a P0 peptide solution at 2 mg / ml, and a Freund's complete adjuvant (Difco Laboratories) was added with heat-killed tuberculosis bacteria H37RA at 20 mg / mL. These two were mixed in equal volumes to prepare an emulsified adjuvant (hereinafter referred to as "P0 (180-199) / CFA").

[0320] The dosage regimen and treatment regimen of the immunization solution are shown in Figure 3 (A). Figure 3 In (A), the first administration day of P0(180-199) / CFA was set as the start day of the experiment (hereinafter referred to as "Day 0"). Pertussis toxin (#180List Biological Laboratories, Figure 3 The administration days of SHED-CM obtained in Example 2 are shown as Day 3 and Day 8 from Day 0. PT represents the administration day of pertussis toxin.

[0321] For each mouse in the negative control group and the treatment group, 50 μL of P0 (180-199) / CFA was subcutaneously injected into the dorsal side between the left hind limb and the tail (day 0), and the same amount was subcutaneously administered again on the opposite side of the first time on day 8. Furthermore, pertussis toxin was intraperitoneally administered to the above mice at 400 ng / mouse on day -1, and 300 ng / mouse on days 1 and 3, respectively, to prepare EAN model mice. The clinical signs of the immunized mice were scored every 2 to 3 days according to the following criteria.

[0322] 0 points: No clinical signs

[0323] 1 point: decreased activity

[0324] 2 points: tail paralysis or mild paralysis of hind limbs

[0325] 3 points: Mild movement disorder or hind limb paralysis

[0326] 4 points: severe movement disorder

[0327] For the EAN model mice prepared above, 200 μL of DMEM or SHED-CM was intraperitoneally administered five times on days 15, 17, 20, 22, and 24. Clinical sign scores were scored according to the above criteria and calculated as mean ± SD. The Student's t-test was used to confirm whether there was a significant difference. p < 0.05 was indicated as *, p < 0.01 was indicated as **, and p < 0.001 was indicated as ***. The results are shown in Figure 3 (B) in.

[0328] In the negative control group, a slow increase in clinical sign scores was also observed after the administration of DMEM (after the 15th day). In contrast, in the treatment group, a downward trend in the scores was observed on the second day (17th day) after the administration of SHED-CM, and a significant decrease in the scores was recorded after the 5th day (20th day) compared with the negative control group ( Figure 3 (B)). These results suggest that SHED-CM has a therapeutic effect on Guillain-Barré syndrome, an autoimmune neuritis.

[0329] (Example 5) Study on the mechanism of action of SHED-CM in the treatment of autoimmune neuritis

[0330] The sciatic nerves of the two groups were collected and subjected to histopathological examination using an optical microscope and an electron microscope and Western blotting to investigate the effect of SHED-CM administration on EAN mice.

[0331] (1) Histopathological examination

[0332] On the 31st day, the mice were euthanized, and the sciatic nerves were collected and fixed with Karnovsky's fixative and epoxy-embedded. The nerves were sliced ​​into 0.5 μm thick sections and stained with toluidine blue. The nerves were observed under an optical microscope ( Figure 4 (A1) and Figure 4 (B1): ×100 times, Figure 4 (A2) and Figure 4 (B2): ×200, manufactured by Thermo Fisher Scientific, product number EVOS Cell Imaging System) and electron microscope ( Figure 4 (A3) and (B3): ×500, manufactured by Japan Electronics Co., Ltd., product number JEM-1400Flash) were observed. The results are shown in Figure 4 In the negative control group, the thickness of myelin formed by Schwann cells of the sciatic nerve was thin, the size of Schwann cells was inconsistent, and there were many small cells ( Figure 4 (A)). In contrast, in the SHED-CM-treated group, it can be seen that there are more Schwann cells with thick nerves and thick myelin sheaths ( Figure 4 (B)). This result means that SHED-CM directly or indirectly acts on the thickness of the myelin sheath, that is, Schwann cells.

[0333] (2) Quantitative PCR and Western Blot

[0334] EAN model mice were prepared by the same method as in Example 4 and divided into a negative control group (4 mice) and a treatment group (5 mice). 200 μL of SHED-CM was intraperitoneally administered 5 times on the 13th, 15th, 17th, 20th and 22nd days, and clinical signs were scored every 2 to 3 days according to the above criteria. On the 23rd day, the mice were euthanized, and the sciatic nerve and cauda equina nerve were collected, and the obtained samples were divided into two equal parts.

[0335] Total RNA was extracted from one of the aliquoted tissues using a total RNA extraction kit (RNeasy Mini Kit, QIAGEN, according to the protocol attached to the kit), and cDNA was prepared using the reverse transcriptase SuperScriptVI from Thermo Fisher Scientific. The expression analysis based on quantitative PCR was performed on the mRNA of each gene of myelin basic protein (MBP), TNF-α, IFN-γ, and IL-17A. The PCR conditions were the same as above. Hypoxanthine-guanine phosphorylation nucleoside transferase (HPRT) was used as an internal standard gene, and its relative ratio was expressed and calculated as mean ± SD. The results are shown in Figure 5 (A) and Figure 6 . Figure 6 In the table, as the result of statistical test, p<0.05 is indicated by *, and p<0.001 is indicated by ***. Figure 6 In the graphs of TNF-α / HPRT and IL-17A / HPRT in the cauda equina in (B), "0.07" indicates the p value when the above statistical processing was performed. In addition, the primer sets used in the quantitative PCR are shown in Table 17 below.

[0336] [Table 17]

[0337]

[0338] In addition, a surfactant 1% Nonidet p40 (NP40) solution containing a protease inhibitor (product name: protease inhibitor cocktail, Sigma Aldrich) and a phosphatase inhibitor (product name: protease / phosphatase inhibitor cocktail, Cell Signaling) was used to prepare a cell lysate from the other aliquot of tissue, and the protein amount was adjusted to a certain amount using the Takara BCA protein detection kit (Takara Bio). Then, the cells were subjected to SDS-PAGE electrophoresis, and protein immunoblotting analysis was performed using anti-MBP antibody, anti-phosphorylated c-met antibody (Cell Signaling), and anti-actin antibody (Santa Cruz). The results are shown in Figure 5 (B) and Figure 7 .

[0339] Depend on Figure 5 As shown in (A), the mRNA expression level of MBP increased by the administration of SHED-CM. Figure 5 (B) shows that the amount of MBP protein also increased after the administration of SHED-CM. MBP is the main protein constituting myelin sheath, and the increase in its amount is consistent with the microscopic observation result of myelin sheath thickening ( Figure 4 ) are consistent.

[0340] In addition, by Figure 6 The quantitative PCR results shown in the figure show that the expression levels of various inflammatory cytokines were reduced in all nerve tissues, and Figure 7 It can be seen that the phosphorylation level of c-Met is increased. It is known that c-Met is a receptor for hepatocyte growth factor (HGF), and it is phosphorylated by receiving HGF. Therefore, the above results suggest that HGF and the like contained in SHED-CM have anti-inflammatory effects and are involved in the improvement of autoimmune neuritis (effectiveness).

[0341] (3) Confirmation of the role of HGF contained in SHED-CM

[0342] In order to investigate whether HGF in SHED-CM is effective in autoimmune neuritis, the same method as in Example 4 was used to Figure 8 EAN model mice were prepared according to the adjuvant and antigen administration schedule shown in (A) and divided into a negative control group, a positive control group 1, a positive control group 2, and a test group (n=5 in each group). Figure 8 The abbreviations in (A) are the same as Figure 3 The statistical processing was performed in the same manner as in Example 4 above.

[0343] During the treatment, DMEM was administered to the negative control group, and SHED-CM was administered to the positive control group 1. In addition, SHED-CM and IgG isotype were administered to the positive control group 2, and SHED-CM and anti-HGF antibody, which is a neutralizing antibody against HGF, were administered to the test group. The results are shown in Figure 8 It should be noted that only the temporal changes in clinical sign scores of the positive control group 2 and the test group are shown.

[0344] In the test group, no improvement in clinical sign scores was observed due to the administration of SHED-CM compared with the positive control 2. This result suggests that at least HGF contained in SHED-CM is involved in the alleviation of symptoms of autoimmune neuritis.

[0345] Based on the above results, SHED-CM enhances myelin damage repair and regeneration and inhibits inflammation, and these factors are believed to contribute to recovery from EAN.

[0346] (Example 6) Effect of SHED-CM on Schwann cells

[0347] In order to investigate in detail the effect of SHED-CM on Schwann cells, an in vitro experiment was conducted using the mouse Schwann cell line IMS32. IMS32 was cultured in a 10 cm culture dish using an IMS32 culture medium (Cosmo Bio) at 37°C in a 5% CO2 incubator until it was 80% confluent. Next, the culture medium was replaced with a serum-free DMEM culture medium in which SHED-CM (1%, 3%, 10% or 30% (v / v)) and either an anti-HGF antibody (1 μg / mL) or an IgG isotype (1 μg / mL) were added. After culturing for 72 hours at 37°C in a 5% CO2 incubator, the proliferation of IMS32 was observed. The results are shown in Fig. 9 As shown in the optical microscope images, IMS32 cultured in a normal serum-free medium (DMEM) was significantly different from that before the experiment ( Fig. 9 (A) Similarly, the cells remain round and barely proliferate ( Fig. 9(B1) and (B2)). In contrast, it was found that when cultured in a medium containing SHED-CM for 72 hours, IMS32 cells showed an elongated morphology and proliferated well ( Fig. 9 of (C1) and (C2)).

[0348] In order to quantify the proliferation degree of IMS32, the ATP production of the cultured IMS32 was measured using the CellTiter-Glo 2.0 cell viability assay system (Promega). As a measuring instrument, a GloMaxDiscover microplate reader GM3000 (Promega) was used. The results are shown in Fig.10 . Fig.10 In the table, N / C indicates a control cultured with DMEM alone. In addition, statistical analysis was performed using one-way ANOVA and Dunnett's test, with p<0.05 indicated by *, p<0.01 indicated by **, and p<0.001 indicated by ***.

[0349] Depend on Fig.10 (A) shows that the proliferation ability of IMS32 was significantly enhanced in a SHED-CM concentration-dependent manner by culturing in a medium containing SHED-CM. In addition, when anti-HGF antibody was added to the medium containing SHED-CM, the proliferation ability of IMS32 was significantly reduced compared with the control antibody ( Fig.10 (B)), it can be seen that part of the proliferation enhancement effect of SHED-CM is at least due to the action of HGF.

[0350] In order to determine whether HGF alone can enhance the proliferation of Schwann cells, the same culture experiment as described above was conducted using IMS32. In this experiment, DMEM medium to which SHED-CM (10%, 30%, 50% (v / v)) or recombinant human (rh) HGF (0.1, 1, 10, 50 ng / mL) (R&D Company) was added was used to culture IMS32 at 37°C in a 5% CO2 incubator for 72 hours. In addition, the amount of ATP produced by IMS32 after culture was quantified in the same manner as described above, and the proliferation ability of IMS32 was investigated using this as an indicator. The results are shown in Fig.11 As Fig.11 In the statistical test results, p<0.05 is indicated as *, and p<0.001 is indicated as ***. In SHED-CM, an enhancement in cell proliferation was observed, whereas no enhancement in proliferation was observed in the medium supplemented with only HGF ( Fig.11 (A)).

[0351] In addition, the same experiment as above was performed with DMEM containing 1% fetal bovine serum (FBS) (Sigma Aldrich). The results are shown in Fig.11 (B) No enhancement of proliferation ability was observed when HGF was used in the absence of FBS, but the proliferation ability of IMS32 was enhanced even when HGF alone was used in the presence of FBS.

[0352] These results suggest that not only HGF but also SHED-CM and an unidentified molecule X contained in serum are essential for the enhancement of the proliferation ability of Schwann cells.

[0353] Next, to understand how SHED-CM and HGF promote the proliferation of Schwann cells, we investigated the intracellular signal transduction pathway. First, SHED-CM and DMEM as a negative control group were concentrated to about 10 times using Amicon Ultra Centrifugal Filter Units 10k (Merck Co., Ltd.) while removing substances with a molecular weight of 10,000 or less. IMS32 (2×10 6 10 cells) were suspended in 50 μL of serum-free DMEM, and then 50 μL of the concentrated medium was added to stimulate the IMS32 cells for 5 minutes. It should be noted that as a positive control, 50 μL of rhHGF prepared to 50 ng / mL using DMEM was also used. Afterwards, the cells stimulated as above were dissolved separately, and these lysates were used for SDS-PAGE and protein immunoblotting.

[0354] Here, as antibodies, anti-phosphorylated c-met antibody, anti-c-met antibody (Sigma Aldrich), anti-phosphorylated Erk1 / 2 antibody (Cell Signaling), anti-Erk1 / 2 antibody (Cell Signaling), anti-phosphorylated AKT antibody (Cell Signaling), and anti-AKT antibody (Cell Signaling) were used. The results are shown in Fig.12 .

[0355] SHED-CM and rhHGF induced the phosphorylation of ERK1 / 2 and AKT. ERK1 / 2 and AKT are located downstream of the HGF receptor c-MET and are important signals for inducing cell proliferation. This suggests that the proliferation and / or regeneration induction of Schwann cells caused by HGF in SHED-CM is important for the alleviation of neuritis symptoms in SHED-CM.

[0356] Next, the proliferation and regeneration of Schwann cells caused by SHED-CM were qualitatively evaluated. Here, IMS32 was cultured using DMEM containing 50% SHED-CM, 50ng / mL rhHGF, 50ng / mL NRG1 (BioLegend), 1mM DBcMP (SigmaAldrich) or 50ng / mL NRG1+1mM DBcAMP, and 5% FBS, and then protein immunoblotting was performed in the same manner as above. In the protein immunoblotting, anti-MBP antibody, anti-p75 antibody (GeneTex), and anti-actin antibody (Santa Cruz) were used. The results are shown in Fig.13 .

[0357] Depend on Fig.13 As shown in the above paragraph, the amount of MBP, a major component of myelin sheath, increased in IMS32 cultured in a medium containing SHED-CM, HGF, and NRG1. Fig.13 As can be seen in the middle of FIG, the amount of p75, a receptor for neurotrophic factors, increased only in IMS32 cells cultured in a medium containing SHED-CM. These results suggest that SHED-CM has the ability to promote the maturation of Schwann cells.

[0358] In addition, since SHED-CM promotes the proliferation of Schwann cells and the regeneration of myelin, it is considered that SHED-CM exerts an effect on all demyelinating diseases. Therefore, the present invention can be used in pharmaceutical preparations for the treatment of demyelinating diseases.

[0359] (Example 7) Study on the mechanism of action of SHED-CM in the treatment of CIPN

[0360] Finally, it was investigated whether symptom improvement was observed in CIPN model mice in which peripheral neuropathy was artificially induced by administration of chemotherapeutic agents by administration of SHED-CM.

[0361] (1) Behavioral pain test

[0362] Peripheral neuropathy was induced by intraperitoneal administration of paclitaxel (80 μg / 200 μL) for a total of 4 times on days 0, 2, 4, and 6. In addition, as a treatment for CIPN, 200 μL of SHED-CM or control culture medium DMEM was intraperitoneally administered on days 1, 3, 5, 7, and for several consecutive days thereafter. In addition, in order to confirm the therapeutic effect, the changes in the escape response to non-noxious stimulation caused by microfibers were compared (von Frey fiber test). Regarding the changes in the escape response, microfibers with a fixed stimulation intensity were used to poke the hind legs for stimulation, and whether the legs were retracted to escape was recorded as 0 or ×, thereby calculating the average value of the minimum stimulation intensity required for escape stimulation in the form of 50% threshold (g) according to the following calculation formula and comparing them.

[0363] The above-mentioned 50% threshold value is expressed by the following formula.

[0364] 50% threshold = (10[X f +kδ]) / 10000

[0365] X f : The pressure strength of the fiber used at the end (Aesthesio (registered trademark); DanMic.Global, Campbell, USA). For the pressure strength of the fiber and the accompanying information, refer to Fig.15 (Quoted from Dinamic Global, LLC explanatory book).

[0366] k: A value determined by the arrangement of 0 / ×. Fig.16 and Fig.17 (Quoted from ["Quantitative assessment of tactile allodynia in the rat paw"; SR Chaplan et al; Journal of Neuroscience Methods; Jul; 53(1):55-63.1994:doi:10.1016 / 0165-0270(94)90144-9.])

[0367] δ: Deviation of fiber (use 0.224)

[0368] The analysis results are shown in Fig.14 The threshold of the negative control mice that were not administered paclitaxel was roughly between 1.0 and 1.5 g, while the CIPN model mice that were administered paclitaxel became sensitive to low stimuli and reacted even to low stimuli below 0.5 g. In contrast, the CIPN model mice that were administered SHED-CM escaped only when the same stimulation as the negative control mice was applied as the administration continued. This result means that the administration of SHED-CM is useful for the relief or treatment of CIPN neuropathies.

[0369] Industrial Applicability

[0370] The present invention is useful in the medical field, particularly in the medical field related to the treatment of neuritis.

[0371] Sequence Listing Free Text

[0372] SEQ ID NO: 1: Base sequence of a DNA fragment containing a gene introduced into a cell (1)

[0373] SEQ ID NO: 2: Base sequence of a DNA fragment containing a gene introduced into a cell (2)

[0374] SEQ ID NO: 3: Base sequence of a DNA fragment containing a gene introduced into a cell (3)

[0375] SEQ ID NO: 4: Base sequence of a DNA fragment containing a gene introduced into a cell (4)

[0376] SEQ ID NO: 5: Base sequence of a DNA fragment containing a gene introduced into a cell (5)

[0377] SEQ ID NO: 6: Base sequence of a DNA fragment containing a gene introduced into a cell (6)

[0378] SEQ ID NO: 7: PCR primer (1)

[0379] SEQ ID NO: 8: PCR primer (2)

[0380] Sequence number 9: PCR primer (3)

[0381] SEQ ID NO: 10: PCR primer (4)

[0382] SEQ ID NO: 11: PCR primer (5)

[0383] SEQ ID NO: 12: PCR primer (6)

[0384] SEQ ID NO: 13: PCR primer (7)

[0385] SEQ ID NO: 14: PCR primer (8)

[0386] SEQ ID NO: 15: PCR primer (9)

[0387] SEQ ID NO: 16: PCR primer (10)

[0388] SEQ ID NO: 17: PCR primer (11)

[0389] SEQ ID NO: 18: PCR primer (12)

[0390] SEQ ID NO: 19: PCR primer (13)

[0391] SEQ ID NO: 20: PCR primer (14)

[0392] SEQ ID NO: 21: PCR primer (15)

[0393] SEQ ID NO: 22: PCR primer (16)

[0394] SEQ ID NO: 23: PCR primer (17)

[0395] SEQ ID NO: 24: PCR primer (18)

[0396] SEQ ID NO: 25: PCR primer (19)

[0397] Sequence number 26: Primer for PCR (20).

Claims

1. A composition capable of inducing the regeneration of myelin sheath or the proliferation of Schwann cells, characterized in that: The composition comprises the culture supernatant of immortalized mesenchymal stem cells into which a combination of two or three genes selected from the group consisting of Bmi-1 gene, HPV16-E6 gene and HPV16-E7 gene and telomerase reverse transcriptase (TERT) gene has been introduced.

2. The composition according to claim 1, characterized in that The gene combination is a combination of Bmi-1 gene, HPV16-E6 gene, HPV16-E7 gene and hTERT gene.

3. The composition according to claim 1 or 2, characterized in that The immortalized mesenchymal stem cells are prepared from dental pulp stem cells.

4. The composition according to claim 3, characterized in that The dental pulp stem cells are human-derived dental pulp stem cells.

5. The composition according to claim 4, characterized in that The method comprises introducing the gene combination into the mesenchymal stem cells to immortalize the mesenchymal stem cells using a viral vector, and culturing the immortalized mesenchymal stem cells in a serum-free medium for 60 to 84 hours to obtain a culture supernatant.

6. The composition according to claim 4, characterized in that It contains at least a component that acts on c-MET, a receptor for hepatocyte growth factor (HGF).

7. A pharmaceutical preparation for treating demyelinating diseases, characterized in that: It contains the composition according to claim 4 as an active ingredient.

8. The pharmaceutical preparation according to claim 7, characterized in that When the total weight of the preparation is set to 100%, the composition is contained in an amount of 10 to 90 weight %.

9. The pharmaceutical preparation according to claim 7, characterized in that The demyelinating disease is a central nervous system disease and is any one selected from the group consisting of multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, atopic myelitis, progressive multifocal leukoencephalopathy, HTLB-1-related myelopathy, HIV-related leukoencephalopathy, subacute sclerosing panencephalitis, osmotic demyelination syndrome and drug-induced leukoencephalopathy.

10. The pharmaceutical preparation according to claim 7, characterized in that The demyelinating disease is a peripheral neuropathy, and is any one selected from the group consisting of Guillain-Barré syndrome, Fisher's syndrome, diabetic neuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, multifocal motor neuropathy, chemotherapeutic agent-induced peripheral neuropathy, and herpes zoster.

11. The pharmaceutical preparation according to claim 7, characterized in that It is any dosage form selected from the group consisting of powder, tablet, capsule, liquid, injection and patch.

12. A pharmaceutical preparation for treating chemotherapeutic agent-induced peripheral neuropathy, characterized in that: The composition contains, as an active ingredient, the culture supernatant of immortalized mesenchymal stem cells into which a combination of two or three genes selected from the group consisting of Bmi-1 gene, HPV16-E6 gene and HPV16-E7 gene and telomerase reverse transcriptase (TERT) gene are inserted.

13. The pharmaceutical preparation according to claim 12, characterized in that The chemotherapeutic agent is an anticancer agent.

14. The pharmaceutical preparation according to claim 13, characterized in that The anticancer agent is a vinca alkaloid preparation or a taxane preparation.

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