Schwann cell and preparation method thereof

Abstract

The present invention aims to provide a method for obtaining Schwann cells directly (by direct reprogramming) without going through pluripotent stem cells such as ES cells or iPS cells. As a means for solving the above-mentioned problems, a method for preparing Schwann cells is provided, which comprises the step of introducing at least one gene selected from the group consisting of SOX10 gene and KROX20 gene or its expression product into somatic cells of a mammal.

Description

[0001] This invention patent application is a divisional application for an application with an application date of September 11, 2015, application number 201580049009.4, and invention name “Schwann cells and preparation methods thereof”. Technical Field

[0002] The present invention mainly relates to a Schwann cell and a preparation method thereof, and in detail, relates to a method for preparing Schwann cells by direct reprogramming. Background Art

[0003] It is believed that Schwann cells play a decisive and important role in the regeneration of nerves. There are also many diseases associated with nerve defects or functional defects of Schwann cells. For these diseases, it is expected that if one's own Schwann cells can be transplanted, it will become an ideal regenerative medicine. In fact, for nerve damage caused by trauma or malignant tumor resection, autologous nerve transplantation, or the treatment of separating and culturing Schwann cells from autologous nerves and transplanting them improves the effect. However, the collection of nerves is very invasive to patients, and secondary nerve damage cannot be avoided in any case. In addition, the number of Schwann cells that can be supplied is mostly insufficient.

[0004] In non-patent literature 1 and 2, the following method is disclosed: using mesenchymal stem cells represented by undifferentiated adipose stem cells (ADSC) (also called adipose-derived stromal cells) as materials, Schwann cell-like cells (dADSC) are differentiated. However, due to the nature of the method, there is a risk of infection from the outside, so there are problems such as difficulty in quality management, cost and manpower. It is also pointed out that the shape or function of the obtained cells is different from that of real Schwann cells. In addition, there is no report that the Schwann cells produced by these methods have the ability to myelinate, and may not contribute to saltatory conduction.

[0005] Recently, it has been shown that cardiomyocytes or hepatocytes can be directly induced (directly reprogrammed or directly converted) from fibroblasts. If Schwann cells can be directly produced from somatic cells such as fibroblasts that can be collected from patients with low invasiveness, the invasiveness will be low and the risk of cancer will also be low, which can be expected to be related to the technology of producing new autologous Schwann cells for transplantation.

[0006] Regarding the genome in which tissue-specific transcription factors are introduced into somatic cells, it is possible to induce differentiation into the tissue cells directly without going through iPS cells (direct reprogramming (direct conversion)), for example, the following reports exist:

[0007] Mouse fibroblasts → chondrocytes (introduced SOX9+Klf4+c-Myc genes)

[0008] Mouse fibroblasts → cardiomyocytes (introduced GATA4+Mef2c+Tbx5 genes)

[0009] Mouse fibroblasts → hepatocytes (Hnf4α+ (introduced Foxa1, Foxa2, or Foxa3) gene)

[0010] Mouse fibroblasts → neural stem cells (introduced with Sox2+FoxG1 genes, etc.), mouse and human cells → hematopoietic stem cells, etc.

[0011] However, there are no reports on the direct conversion of somatic cells into Schwann cells.

[0012] Prior art literature

[0013] Non-patent literature

[0014] Non-patent document 1: Kingham PJ, DF Kalbermatten, D Mahay, et al: Adipose-derivedstem cells differentiate into a Schwann cell phenotype and promote neuriteoutgrowth in vitro. ExpNeurol, 2007; 207:267-274.

[0015] Non-patent literature 2: Liu Y, Zhang Z, Qin Y, Wu H, Lv Q, Chen X, Deng W: A new method for Schwann-like cell differentiation of adipose derived stem cells. NeurosciLett. 2013 Sep 13; 551:79-83. Summary of the invention

[0016] Problems to be solved by the invention

[0017] An object of the present invention is to provide a method for preparing Schwann cells with a low risk of canceration, which can be applied to the treatment of diseases associated with nerve defects or functional defects of Schwann cells.

[0018] Technical solutions to solve problems

[0019] The present inventors have found that Schwann cells can be obtained directly (by direct reprogramming) without going through pluripotent stem cells such as ES cells or iPS cells by combining and introducing specific genes into somatic cells of mammals.

[0020] The present invention includes the following aspects.

[0021] Item 1. A method for producing Schwann cells, comprising the step of introducing at least one gene selected from the group consisting of SOX10 gene and KROX20 gene or an expression product thereof into somatic cells of a mammal.

[0022] Item 2. The method according to Item 1, wherein the gene is a combination of the SOX10 gene and the KROX20 gene.

[0023] Item 3. The method according to Item 1 or 2, wherein the somatic cells are fibroblasts, vascular endothelial cells or mesenchymal stem cells.

[0024] Item 4. A Schwann cell derived from a somatic cell of a mammal, comprising at least one gene selected from the group consisting of exogenous SOX10 gene and KROX20 gene or an expression product thereof.

[0025] Item 5. A transplant material for treating a disease based on a defect in nerves or a defect, deficiency or reduced function of Schwann cells, comprising cells obtained by the method of any one of Items 1 to 3, or the Schwann cells of Item 4.

[0026] Item 6. A composition for preparing Schwann cells, comprising at least one gene selected from the group consisting of SOX10 gene and KROX20 gene or an expression product thereof.

[0027] Effects of the Invention

[0028] In the present invention, Schwann cells can be provided from somatic cells in a short period of time by direct reprogramming. The Schwann cells can be easily induced from the somatic cells of the person to be transplanted, so when the obtained Schwann cells are transplanted, there is no problem of immunological rejection response. In addition, Schwann cells can be induced directly from somatic cells without passing through iPS cells or ES cells, so problems such as canceration caused by pluripotent stem cells can be avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 An outline of an example of the method of the present invention is shown below.

[0030] Figure 2 Representative S100β staining images are shown.

[0031] Figure 3 A typical example of the four stages obtained by staining of S100β is shown.

[0032] Figure 4A Evaluation results based on cell morphology are shown. Typical examples of cell morphology of HDF, cSC, and dSC are shown.

[0033] Figure 4B Indicates the evaluation results based on cell morphology.

[0034] Figure 5A An example of immunostaining of a Schwann cell-associated marker (p75NTR) is shown.

[0035] Figure 5B An example of immunostaining for a Schwann cell-associated marker (GFAP) is shown.

[0036] Figure 5C An example of immunostaining of a Schwann cell-associated marker (Nestin) is shown.

[0037] Figure 5D An example of immunostaining of a Schwann cell-associated marker (NG2) is shown.

[0038] Figure 6 The graph shows the results of measuring the mRNA expression levels of S100β and p75NTR genes.

[0039] Fig. 7A The results of evaluating the effect of elongating neurites on neurons are shown. An example of fluorescently labeled NG108-15 neurons co-cultured with HDF fibroblasts, normal Schwann cells (cSC, positive control), and dSCs is shown. Arrows indicate elongated neurites.

[0040] Figure 7B The results of evaluation of the effect on neurite extension of nerve cells are shown.

[0041] Figure 8 This figure shows an example of immunostaining of markers associated with Schwann cells (S100β, p75NTR, GAP43) during transformation induced by plasmid introduction (electroporation).

[0042] Fig. 9A Phase contrast images of normal human adipose-derived stem cells (ADSCs) before induction, and phase contrast images and S100β staining images after induction of Schwann cells are shown. Magnification 200 times.

[0043] Fig. 9B An example of immunostaining of Schwann cell-associated markers (S100β, GAP43, p75NTR, Protein Zero (PO)) is shown. Magnification 100 times.

[0044] Fig. 10APhase contrast images of umbilical cord vascular endothelial cells (Huvec) before induction, and phase contrast images of Schwann cells after induction and S100β staining images. 200 times.

[0045] Fig. 10B An example of immunostaining images of Schwann cell-associated markers (S100β, GAP43, p75NTR) is shown. Magnification: 100 times.

[0046] Fig.11A An example of an immunostaining image of a myelin marker (Protein Zero (P0)) is shown.

[0047] Fig. 11B An example of an immunostaining image of a myelin marker (Myelin basic protein (MBP)) is shown.

[0048] Fig. 12A The outline of the evaluation test using the sciatic nerve injury model is shown.

[0049] Fig. 12B An example of an immunostaining image using a Schwann cell-associated marker is shown.

[0050] Fig. 12C An example of an immunostaining image using a Schwann cell-associated marker is shown.

[0051] Fig.13A The figure shows the outline of the dSC transplantation experiment in the sciatic defect model of immunodeficient mice.

[0052] Fig. 13B A macroscopic image showing a reconstructed nerve.

[0053] Fig. 13C The myelin staining image of the transverse section of the regenerated nerve tissue is shown.

[0054] Fig.13D It indicates the evaluation results of the Sciatic Function Index (SFI).

[0055] Fig.13E Indicates the evaluation results of innervated muscle atrophy and fibrosis.

[0056] Fig.14 Indicates the production of neurotrophic factors (BDNF, GDNF, NGF). Measured by ELISA. *p<0.05vs.Control; **p<0.01vs.Control; #p<0.05vs.cSC.

[0057] To explain, Figure 2 , Figure 3 , Figure 4A , Figure 5A ~D, Fig. 7A , Figure 8 , Fig. 9A and B. Fig. 10A and B. Fig.11A and B, and Fig. 12B ~C also represents a color-inverted image. DETAILED DESCRIPTION

[0058] The present invention relates to a method for preparing Schwann cells, which is a method for preparing Schwann cells without using pluripotent stem cells such as ES cells or iPS cells.

[0059] Schwann cells

[0060] Schwann cells are glial cells in the peripheral nervous system. Under physiological conditions, they support nerve tissue, form myelin (myelin sheath) and help achieve saltatory conduction. When peripheral nerves are damaged, they complete the production and release of neurotrophic factors, the basis for regenerating axons, myelin formation and many other important functions in peripheral nerve regeneration.

[0061] Natural Schwann cells are different from nerve cells that are directly derived from the ectoderm, and are derived from the neural crest. Mature Schwann cells are formed through Schwann precursor cells and immature Schwann cells. In this specification, for simplicity, all cells in the middle of these differentiation processes are included in "Schwann cells".

[0062] In addition, among Schwann cells, there are Schwann cells that form myelin, and motile Schwann cells that do not form myelin (undifferentiated Schwann cells), and the like, and all of these are included in the "Schwann cells" in this specification.

[0063] In the present specification, not only cells identical to natural Schwann cells but also cells whose functions can be regarded as partially or completely identical to those of natural Schwann cells (may also be referred to as "Schwann cell-like cells") are referred to as "Schwann cells".

[0064] Somatic cells

[0065] Somatic cells can be derived from mammals. Particularly preferably, cells derived from mammals, and not Schwann cells themselves and cells with the ability to differentiate into Schwann cells in vivo are used. In the case of transplanting Schwann cells into an organism, in order to reduce the risk of infection or rejection response, it is preferred to use somatic cells (self cells) derived from the subject to be transplanted. However, in the case of transplantation for purposes such as sudden nerve damage, Schwann cells prepared in advance from somatic cells of other people or other animals that are not self cells can be used for transplantation. Or Schwann cells can be made from somatic cells of other people or other animals prepared in advance for transplantation. That is, a library of Schwann cell banks (a library containing Schwann cell precursor cells) can be made and provided for transplantation purposes. In this case, in order to reduce the risk of rejection response, MHC can be typed in advance. In addition, the cell characteristics or tumorigenicity of Schwann cells can be confirmed in advance.

[0066] In the present specification, examples of mammals include mice, rats, hamsters, humans, dogs, cats, monkeys, rabbits, cows, horses, pigs, and the like, and particularly include humans.

[0067] The somatic cells to be used as the subject of the method (direct reprogramming) of the present invention are not particularly limited.

[0068] Somatic cells can use somatic cells that can be easily collected from organisms. For example, fibroblasts, keratinocytes, oral mucosal epithelial cells, nasal mucosal epithelial cells, tracheal mucosal epithelial cells, gastric mucosal epithelial cells, intestinal mucosal epithelial cells, vascular endothelial cells, smooth muscle cells, adipocytes, gingival cells (gingival fibroblasts, gingival epithelial cells), medullary cells, periodontal membrane cells, medullary cells, medullary cells, medullary mesenchymal cells, leukocytes, lymphocytes, myocytes, conjunctival epithelial cells, osteoclasts, etc., preferably fibroblasts, keratinocytes, oral mucosal epithelial cells, gingival cells, leukocytes, lymphocytes, etc. are listed. In the present invention, it is preferred to use the above-mentioned cells collected from organisms.

[0069] "Organism" includes not only embryos (fetuses), larvae, young bodies, and adults, but also placentas and umbilical cords that connect mothers and fetuses. Umbilical cord vascular endothelial cells and other cells derived from the umbilical cord or cells derived from the placenta are not strictly somatic cells, but these cells are also included in the "somatic cells" of the present invention (in this case, "somatic cells" are replaced with another word and read as "umbilical cord vascular endothelial cells", "cells derived from the umbilical cord", "cells derived from the placenta", etc.). From the perspective of ease of collection, these cells are an example of preferred somatic cells.

[0070] In addition, somatic cells produced by induction of differentiation, dedifferentiation, or reprogramming of somatic stem cells such as mesenchymal stem cells (MSC), neural stem cells, hepatic stem cells, intestinal stem cells, skin stem cells, hair stem cells, pigment cell stem cells, etc. can also be listed. In addition, cells induced to differentiate, dedifferentiate, or reprogram to be induced into other somatic cells by various somatic cells can also be listed. In addition, somatic cells induced to differentiate, dedifferentiate, or reprogram by cells of the germ line can also be listed.

[0071] In addition, somatic cells induced by differentiation or reprogramming of embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) can also be mentioned.

[0072] In addition, in addition to the above-mentioned differentiated somatic cells, somatic stem cells can also be used. Here, stem cells refer to cells with the ability to self-replicate and differentiate into other types of cells. Specifically, examples include somatic stem cells such as mesenchymal stem cells (MSC) (e.g., adipose derived stromal cells (ADSC) derived from fat), neural stem cells, hepatic stem cells, intestinal stem cells, skin stem cells, hair stem cells, and pigment cell stem cells.

[0073] In addition, strictly speaking, ES cells, iPS cells, or germ line cells are not somatic cells and are also included in the "somatic cells" of the present invention (in this case, "somatic cells" are replaced with another word such as "ES cells", "iPS cells" or "germ line cells").

[0074] In addition, cultured cells can also be listed, and somatic cells induced by differentiation, dedifferentiation, or reprogramming can also be listed. In addition, somatic cells induced by differentiation, dedifferentiation, or reprogramming by ES cells, iPS cells, or germline cells can also be listed.

[0075] In a preferred embodiment of the present invention, the somatic cells are fibroblasts, vascular endothelial cells (particularly umbilical cord vascular endothelial cells), or mesenchymal stem cells (particularly adipose-derived stromal cells).

[0076] Gene or its expression product

[0077] In the method of the present invention, at least one gene selected from the group consisting of SOX10 gene and KROX20 gene or its expression product is introduced into somatic cells. Here, examples of the "expression product" include mRNA or protein of SOX10 gene and / or KROX20 gene.

[0078] The gene combination that can be used includes only the SOX10 gene, only the KROX20 gene, and a combination of the SOX10 gene and the KROX20 gene. From the viewpoint of the efficiency of obtaining Schwann cells, a combination of the SOX10 gene and the KROX20 gene is preferred.

[0079] In the method of the present invention, genes other than one or two of the SOX10 gene and / or the KROX20 gene may also be used simultaneously. In addition, microRNA, siRNA, shRNA or DNA expressing them may also be used simultaneously. In addition, various proteins may also be used simultaneously. In addition, various other genes may also be introduced simultaneously. From the viewpoint of the efficiency of obtaining Schwann cells and the viewpoint of simplicity, it is preferred to use only one or two genes, particularly two genes of the SOX10 gene and the KROX20 gene.

[0080] The SOX10 gene encodes a transcription factor belonging to the SOX (SRY-related HMG-box) family that is involved in the control of cell fate determination during embryogenesis.

[0081] The KROX20 gene (alias, EGR2, AT591, CMT1D, CMT4E) encodes a protein with three C2H2-type zinc fingers.

[0082] The above genes are all highly conserved genes in vertebrates, and in this specification, unless the animal name is particularly shown, it means that the genes of the same family are included. In addition, it also includes genes containing polymorphism, even if they are genes with mutations, they have the same function as the wild-type gene product.

[0083] For example, the cDNA base sequences of the SOX10 gene and KROX20 gene of humans (Homo sapiens) and mice (Mus musculus) and the amino acid sequences of the proteins encoded by them are registered in GenBank provided by the National Center for Biotechnology Information (NCBI) with the following accession numbers (when multiple revisions are registered, it is understood that it refers to the latest revision):

[0084] Human SOX10 gene cDNA sequence: NM_006941 (e.g. NM_006941.3),

[0085] Human SOX10 protein amino acid sequence: NP_008872 (e.g., NP_008872.1);

[0086] Mouse Sox10 gene cDNA sequence: NM_011437 (e.g. NM_011437.1),

[0087] Mouse SOX10 protein amino acid sequence: NP_035567 (e.g., NP_035567.1);

[0088] Human KROX20 gene cDNA sequences: NM_000399, NM_001136177, NM_001136178, NM_001136179 (e.g., NM_000399.3, NM_001136177.1, NM_001136178.1, NM_001136179.1),

[0089] Human KROX20 protein amino acid sequence: NP_000390, NP_001129649, NP_001129650, NP_001129651 (e.g., NP_000390.2, NP_001129649.1, NP_001129650.1, NP_001129651.1);

[0090] Mouse Krox20 gene cDNA sequence: NM_010118 (e.g. NM_010118.3)

[0091] Mouse KROX20 protein amino acid sequence: NP_034248 (eg, NP_034248.2).

[0092] Import

[0093] The method of the present invention can be carried out according to a known direct reprogramming method, for example, according to any of the following methods, except for selecting a specific gene and using a culture medium suitable for Schwann cells:

[0094] 1: Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors; Masaki Ieda, Ji-Dong Fu, Paul Delgado-Olguin, Vasanth Vedantham, Yohei Hayashi, Benoit G. Bruneau, and Deepak Srivastava Cell142:375-386, 2010.

[0095] 2: Direct conversion of fibroblasts to functional neurons by defined factors. Thomas Vierbuchen, Austin Ostermeier, Zhiping P. Pang, Yuko Kokubu, Thomas C. Sudhof & Marius Wernig. Nature 463:1035-1041, 2010

[0096] 3:Induction of human neuronal cells by defined transcription factors. Pang ZP, Yang N, Vierbuchen T, Ostermeier A, Fuentes DR, Yang TQ, Citri A, Sebastiano V, Marro S, Sudhof TC, Wernig M. Nature 476:220-223, 2011.

[0097] 4:Generation of hyaline cartilaginous tissue from mouse adult dermalfibroblast culture by defined factors Kunihiko Hiramatsu,Satoru Sasagawa,Hidetatsu Outan

[0098] i, Kanako Nakagawa, Hideki Yoshikawa, and Noriyuki Tsumaki, Journal of Clinical Investigation, 121:640-657, 2011.

[0099] 5:Induction of functional hepatocyte-like cells from mousefibroblasts by defined factors.Pengyu Huang,Zhiying He,Shuyi Ji,Huawang Sun,Dao Xiang,Changcheng Liu,Yiping Hu,XinWang&Lijian Hui,.Nature 475:386-389,2011.

[0100] 6: Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Sayaka Sekiya&Atsushi Suzuki. Nature 475:390-393, 2011.

[0101] 7: International Publication No. WO2014 / 010746

[0102] The contents of the above-mentioned Documents 1 to 7 are incorporated herein by reference.

[0103] Specifically, it is preferred that the target gene is inserted into one or more expression vectors, the expression vectors are introduced into target somatic cells, and the gene is expressed in the cells.

[0104] As the method for introducing gene, the method of infecting viral vectors such as retroviral vector, adenoviral vector, slow virus vector, adeno-associated virus vector, herpes virus vector, Sendai virus vector can be enumerated. In addition, when introducing gene and its expression product, the method of transfecting the expression product (mRNA, protein) of plasmid vector or additional vector, gene can also be carried out by non-viral vectors such as cationic liposome, cationic polymer, electroporation. In addition, mRNA can also be introduced. These means for gene introduction are all included, and are referred to as carriers in this manual.

[0105] From the viewpoint of introduction efficiency and stable maintenance of introduced genes, viral vectors are preferred, and in order to suppress the risk of carcinogenesis, plasmids are preferred.

[0106] Alternatively, by introducing a gene serving as a drug selection marker (puromycin resistance, blasticidin S resistance, neomycin resistance, hygromycin resistance, etc.) together with the target gene, followed by drug selection, cells expressing the target gene can be selected and used.

[0107] The gene of the present invention can be introduced via a plasmid, or a viral vector such as a retroviral vector can be used. From the viewpoint of introduction efficiency and stable maintenance of the introduced gene, a viral vector is preferred, and in order to suppress the risk of canceration, a plasmid is preferred.

[0108] The gene introduced into the somatic cell can also be transcribed by the LTR promoter, or can be expressed by other promoters in the vector. For example, constitutive expression promoters such as CMV promoter, EF-1α promoter, CAG promoter, or desired inducible promoters can be used. In addition, a chimeric promoter in which a part of the LTR is replaced by another promoter can be used.

[0109] When the introduced factor is an expression product of a gene (eg, a protein), the protein as the expression product can be introduced into somatic cells by binding a peptide called Protein Transduction Domain (PTD) or the like to the protein and adding the resultant to the culture medium.

[0110] In one embodiment of the method of the present invention, after the gene or the like is introduced into the somatic cells, the introduced cells can be cultured in a medium suitable for the culture of Schwann cells. As the medium suitable for the culture of Schwann cells, a known medium can be used. For example, a medium (Schwann cell induction medium) can be used which contains one or more (preferably all) of the following components: about 1 to 20 μM (particularly about 5 μM) forskolin; about 2 to 50 ng / ml (particularly about 10 ng / ml) bFGF (basic fibroblast growth factor); about 2 to 50 ng / ml (particularly about 10 ng / ml) PDGF (Platelet-Derived Growth Factor); about 50 to 1000 ng / ml (particularly about 200 ng / ml) human neuregulin-β1 (also known as heregulin, GGF (Glial growth factor)) in a common culture medium such as DMEM medium (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS (fetal bovine serum) (all the above concentrations are final concentrations). As an example, the culture medium described in Non-Patent Document 1 or 2 (a culture medium capable of inducing Schwann cells from undifferentiated adipose stem cells) can be used.

[0111] The culture period is not particularly limited, and can be, for example, about 12 hours to about 1 month, about 1 day to about 3 weeks, or about 3 days to about 2 weeks. The culture medium can be exchanged as needed. The culture conditions are preferably in accordance with conventional methods.

[0112] preparation

[0113] In this way, Schwann cells are induced from somatic cells to prepare Schwann cells.

[0114] The prepared Schwann cells have at least one gene or its expression product selected from the group consisting of exogenous SOX10 gene and KROX20 gene in a certain manner. Here, "exogenous" refers to a manner in which the gene or its expression product is introduced mainly as a result of the above-mentioned introduction means, and a manner different from the natural manner. Examples include genes whose expression is controlled by promoters other than natural promoters, positions on chromosomes other than natural ones, or manners in which genes are present outside chromosomes.

[0115] Now that Schwann cells have been identified, examples of evaluation of Schwann cell function include: observation and evaluation of morphology (e.g., ratio of cell width to cell length); detection of expression of Schwann cell-specific markers such as S100β, p75NTR, GFAP, Nestin, NG2, etc. (e.g., detection of marker gene expression using RT-PCR, detection of marker protein expression using immunostaining, etc.); production of neurotrophic factors; effects on neurite elongation or myelin-forming ability of co-cultured neurons, etc.

[0116] Schwann cells typically have a bipolar or multipolar cell morphology with a relatively small nucleus.

[0117] Among the Schwann cell-specific markers, p75NTR is a marker for undifferentiated Schwann cells.

[0118] The formation of myelin can be confirmed by detecting myelin cell markers such as Myelin Protein Zero (MPZ, P0) and myelin basic protein (MBP), observing the morphology of myelin, and the like.

[0119] Schwann cells are sometimes obtained as a mixture with cells other than Schwann cells (e.g., original somatic cells). In this case, Schwann cells and cells other than Schwann cells can be separated as needed. The means for separation are not particularly limited. For example, when the obtained Schwann cells and fibroblasts as the original cells are separated, they can be separated based on the difference in adhesion of the cells to the base (e.g., collagen, etc.). In general, Schwann cells have weaker adhesion to the base than fibroblasts. In addition, Schwann cells and cells other than Schwann cells can also be separated according to classification.

[0120] The Schwann cells prepared by the present invention can be preferably used as a transplant material described below, for example.

[0121] The Schwann cells prepared by the present invention can also be used for various research and technology development using Schwann cells, etc. For example, it is useful for basic research such as analysis of the mechanism of Schwann cell generation, differentiation, and morphogenesis, and the effects of mechanical stress, nutrition, hormones, etc. on these.

[0122] If the Schwann cells prepared by the present invention are used, Schwann cells can be easily, quickly and cheaply established from humans or animals with various diseases or genetic backgrounds. Therefore, abnormalities of Schwann cells associated with diseases or genetic backgrounds can be analyzed by biochemistry, molecular biology, immunology and other methods, which can be useful for research such as elucidation of the pathogenesis of diseases or development of diagnostic methods. In addition, if such Schwann cells are used for the development of drugs, toxicity tests of drugs, etc., it can be useful for the development of new therapeutic methods for various diseases.

[0123] Transplant materials

[0124] The Schwann cells obtained by the present invention can be used to treat various diseases. In this case, the Schwann cells can be provided in the form of a transplant material.

[0125] Transplant material refers to a material containing Schwann cells that is introduced into a living body for the purpose of repairing and rebuilding nerve fibers. The Schwann cells obtained in the present invention can be used for the preparation of transplant materials. Schwann cells themselves also become transplant materials. Therefore, Schwann cells can also be transplanted into patients as cell preparations, and can be transplanted together with a substrate (scaffold) composed of artificial materials, or transplanted after being cultured together with a scaffold. The substrate (scaffold) acts, for example, as nerve reconstruction. In these cases, the scaffold can be made into various 3D shapes according to the purpose of transplantation.

[0126] The transplant material of the present invention can be produced by a method comprising the above-mentioned method for producing Schwann cells as a step.

[0127] Specific examples of the substrate (scaffold) include a tube made of polyglycolic acid (PGA), a collagen tube, a fibrin glue, a polymer foam tube, a gelatin tube, a tube made of polyglycolic acid (PGA) and collagen, etc. As a tube made of polyglycolic acid (PGA) and collagen, a commercially available product such as "Nabu Brizzi" (manufactured by Toyobo Co., Ltd.) can also be used.

[0128] The transplant material can be used in the treatment of autologous nerve transplantation or transplantation of Schwann cells isolated and cultured from autologous nerves. This method is described in the following literature:

[0129] 1:Hadlock T,Sundback C,Hunter D,Cheney M,Vacanti JP.A polymer foamconduit seeded with Schwann cells promotes guided peripheral nerveregeneration.Tissue Eng 2000;6:119-127.

[0130] 2:Jesuraj NJ,Santosa KB,Macewan MR,Moore AM,Kasukurthi R,Ray WZ,FlaggER,Hunter DA,Borschel GH,Johnson PJ,Mackinnon SE,Sakiyama-Elbert SE.Schwanncells seeded in acellular nerve grafts improve functional recovery.MuscleNerve.2014 Feb;49(2):267-76.

[0131] 3:Tabesh H,Amoabediny G,Nik NS,Heydari M,Yosefifard M,Siadat SO,Mottaghy K.The role of biodegradable engineered scaffolds seeded with Schwanncells for spinal cord regeneration.Neurochem Int.2009 Feb;54(2):73-83.

[0132] 4:Novikova LN,Pettersson J,Brohlin M,Wiberg M,NovikovLN.Biodegradable poly-beta-hydroxybutyrate scaffold seeded with Schwann cellsto promote spinal cord repair.Biomaterials.2008 Mar;29(9):1198-206.

[0133] 5:Guest J,Santamaria AJ,Benavides FD.Clinical translation ofautologous Schwann cell transplantation for the treatment of spinal cordinjury.Curr Opin Organ Transplant.2013Dec;18(6):682-9.

[0134] 6:Brook GA,Lawrence JM,Shah B,Raisman G.Extrusion transplantation ofSchwann cells into the adult rat thalamus induces directional host axongrowth.Exp Neurol.1994Mar;126(1):31-43.

[0135] 7:Vaudano E,Campbell G,Hunt SP.Change in the molecular phenotype ofSchwann cells upon transplantation into the central nervous system:down-regulation of c-jun.Neuroscience.1996Sep;74(2):553-65.

[0136] 8:Keirstead HS,Ben-Hur T,Rogister B,O'Leary MT,Dubois-Dalcq M,Blakemore WF.Polysialylated neural cell adhesion molecule-positive CNSprecursors generate both oligodendrocytes and Schwann cells to remyelinatethe CNS after transplantation.J Neurosci.1999Sep 1;19(17):7529-36.

[0137] 9: Wan H, An YH, Sun MZ, Zhang YZ, Wang ZC. Schwann cells transplantation promoted and the repair of brain stem injury in rats. Biomed Environ Sci. 2003 Sep; 16 (3): 212-8.

[0138] 10: Chen L, Fan

[0139] 11: Shields SA, Blakemore WF, Franklin RJ. Schwann cell remyelination isrestricted to astrocyte-deficient areas after transplantation into demyelinated adult rat brain. J Neurosci Res. 2000 Jun 1;60(5):571-8.

[0140] The contents of the above-mentioned Documents 1 to 11 are incorporated herein by reference.

[0141] Examples of diseases that are the subject of the above-mentioned treatment include: central nervous system defects or damages caused by cerebral infarction, spinal cord injury, etc., and peripheral nervous system defects or damages associated with trauma, neuritis, or tumor resection; central nervous system diseases such as multiple sclerosis, neuromyelitis optica (Devic syndrome), concentric sclerosis (Balo disease), acute disseminated encephalomyelitis (ADEM), inflammatory extensive sclerosis (Schilder's disease), subacute sclerosing panencephalitis (SSPE), and progressive multifocal leukoencephalopathy (PML); peripheral nervous system diseases such as Guillain-Barre syndrome, Fisher syndrome, and chronic inflammatory demyelinating polyradiculoneuritis; and diseases based on Schwann cell defects, insufficiency, or decreased function such as Charcot-Marie-Tooth disease (CMT).

[0142] In this specification, unless otherwise specified, the term "treatment" refers to treatment performed during a patient's period of suffering from a specific disease or disorder, and refers to reducing the severity of the disease or disorder, or one or more of its symptoms, or delaying or slowing down the progression of the disease or disorder. In this specification, "treatment" includes "prevention".

[0143] The Schwann cells obtained in the present invention are not limited to the treatment of diseases, and can also be used for the purpose of beauty or functional enhancement. In this case, the treatment of humans is also conveniently referred to as treatment in this specification, and "patient" can be read as "healthy person" or "human" instead of another word, and "disease" can be read as "beauty" or "function" instead of another word.

[0144] The present invention can be used not only for the treatment of human diseases, but also for the treatment of diseases of mammals including pet animals such as dogs and cats or domestic animals such as cattle, horses, pigs, sheep, and chickens. In this case, "patient" is replaced with another word or phrase such as "patient animal" or "mammal".

[0145] Composition

[0146] As described above, Schwann cells can be prepared by introducing at least one gene or its expression product selected from the group consisting of SOX10 gene and KROX20 gene into somatic cells. Therefore, the present invention further provides a composition for preparing Schwann cells, which comprises at least one gene or its expression product selected from the group consisting of SOX10 gene and KROX20 gene. The composition for preparing the Schwann cells comprises factors used to induce Schwann cells from somatic cells, and the above-mentioned genes or their expression products are preferably contained in a form that can be introduced into somatic cells. The above-mentioned genes are in a form that can be introduced into somatic cells, and specifically, a vector into which the above-mentioned genes are inserted can be exemplified. Here, the above-mentioned genes can be respectively inserted into different vectors, or two or more genes can be simultaneously inserted into one vector.

[0147] The types of carriers that can be used are as described above.

[0148] Direct reprogramming in vivo

[0149] As described above, Schwann cells can be prepared by introducing at least one gene selected from the group consisting of SOX10 gene and KROX20 gene or an expression product thereof into somatic cells.

[0150] Here, when nerves are injured, fibroblasts gather at the injured part, and the gathered fibroblasts subsequently form fibrous scars.

[0151] Therefore, by applying the preparation method of the present invention, at least one gene selected from the group consisting of SOX10 gene and KROX20 gene or its expression product is introduced into fibroblasts of the damaged part of the nerve, Schwann cells are induced by direct reprogramming in the damaged part, thereby contributing to the treatment of nerve damage or nerve regeneration. In the introduction of at least one gene selected from the group consisting of SOX10 gene and KROX20 gene or its expression product, the above-mentioned composition of the present invention can be preferably used.

[0152] The preparation method of the present invention is understood to include direct reprogramming in vivo as described above, in addition to direct reprogramming in vitro.

[0153] In vivo direct reprogramming can be performed by, for example, direct reprogramming of cardiomyocytes in vivo as described in the following literature, in addition to introducing at least one gene selected from the group consisting of SOX10 gene and KROX20 gene or its expression product into fibroblasts of the damaged part of the nerve.

[0154] 1:Ieda M.Heart regeneration using reprogramming technology.Proc JpnAcad Ser B Phys Biol Sci.2013;89(3):118-28.Review.

[0155] 2: Ieda M, Fu JD, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, Srivastava D. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell. 2010 Aug 6;142(3):375-86.

[0156] 3: Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, Liu L, Conway SJ, FuJD, Srivastava D. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature. 2012 May 31;485(7400):593-8.

[0157] The contents of the above-mentioned Documents 1 to 3 are incorporated herein by reference.

[0158] [Example]

[0159] Hereinafter, although an Example is shown, this invention is not limited to this Example.

[0160] In the examples, HDF means human dermal fibroblasts, cSC means cultured Schwann cells collected from a control organism, and dSC means directly reprogrammed Schwann cells obtained by the method of the present invention.

[0161] Cont stands for control.

[0162] Example 1 Overview of the method ( Figure 1 )

[0163] Figure 1The schematic diagram of the method for preparing Schwann cells of the present invention (in the figure, "directly reprogrammed Schwanncell: dSC") is shown.

[0164] The cDNA coding sequences of various genes such as SOX10 were inserted into the retroviral vector plasmid pMXs.puro using the Gene art system. The packaging cells Plat GP were suspended in 1% NEAA 10% FBS DMEM (normal culture medium) containing 100 U / mL penicillin and 100 μg / ml streptomycin, and 5×10 cells were plated in a gelatin-coated 10 cm culture dish. 6 The cells were seeded at a concentration of 100 (Day -3). After 24 hours of culture, pMXs vectors containing various genes were introduced in various combinations together with pCMV VSV vectors using X-tremeGENE 9 at the following ratios. A mixture of 5 μg of the introduced gene, 2.5 μg of pCMV.VSV, 500 μl of Opti-MEM, and 22.5 μl of X-tremeGENE 9 was added to a 10 cm dish filled with 10 ml of culture medium (Day -2). After 24 hours, the culture medium was replaced with a normal culture medium without antibiotics (Day -1). On the same day (Day -1), normal human skin fibroblast cell line (aHDF) (in the figure, "fibroblast") was cultured at a rate of 1.5×10 4 ~2×10 4cells / mL was seeded in a culture dish or 12-well plate. After 24 hours (day 0), the Plat GP culture supernatant was passed through a syringe filter with a pore diameter of 0.45μm and mixed with polybutene (final concentration 4μg / mL) (virus solution). After the aHDF culture supernatant was removed by suction, 1mL of the above virus solution was quickly added and cultured for 24 hours (infection; in the figure, "transfection"). As a control group, cells that were not infected with the virus were also prepared. One day later (Day 1), the culture supernatant was removed by suction, and Schwann cell induction medium was added (usually a medium containing 5mM forskolin, 10ng / ml recombinant human basic fibroblastgrowthfactor(bFGF), 5ng / ml recombinant human platelet-derived growth factor(PDGF)and 200ng / ml recombinant human heregulin1-b1(GGF) (all final concentrations)), and then exchanged with a new culture medium of the same composition every 2 days. On Days 12-22, the obtained cells were stained for S100β, a representative marker of Schwann cells. Cells that were not infected with retroviral vectors and cultured in the same manner were set as Control.

[0165] Example 2 Transformation of normal human skin fibroblasts into Schwann cells, S100β fluorescent immunostaining images (Table 1) Figure 2 )

[0166] Normal human skin fibroblasts (aHDF) were cultured on a 12-well plate and the operations described in Example 1 were performed. On the 14th day, the culture medium was removed by aspiration from each well, washed once with PBS, and then fixed with 4% PFA. A blocking solution was added and allowed to stand at 37°C for 15 minutes. The cells were stained with anti-S100β antibody (primary antibody) and AlexaFluor566-labeled anti-rabbit IgG antibody (secondary antibody). Different gene combinations were introduced into each well of the plate, and which gene combination was introduced into which numbered well is recorded in Table 1 (in the table, there is a substance recorded as "1" in the column of each gene, indicating that the retroviral vector containing the gene is infected, and a blank column indicates that the retroviral vector containing the gene is not infected). Candidate genes for the introduction of SOX10, Krox20, and Oct6, which are Schwann cell-associated genes, are 7 factors of SOX2, C-myc, KLF4, and Oct3 / 4, which are reprogramming-associated genes. For example, No. 43 in Table 1 is a cell infected with a retroviral vector containing genes of SOX10, Krox20, Oct6, and KLF4.

[0167] As a representative staining image of S100β, cells into which genes of two factors, Sox10 and Krox20, were introduced are shown. Figure 2 . Figure 2 Co-staining with nuclear staining caused by DAPI (4',6-diamidino-2-phenylindole).

[0168]

Table 1

[0169] NO. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 evaluate + + - +++ + ++ + + + + +++ +++ ++ ++ ++ ++ ++ ++ SOX10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 KROX20 1 1 1 1 1 1 1 1 1 1 1 OCT6 1 1 1 SOX2 1 1 1 1 c-myc 1 1 1 1 1 KLF4 1 1 1 1 OCT3 / 4 1 1 1 1

[0170] NO. 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 evaluate + ++ ++ ++ +++ ++ ++ + + + + + + + + + + + SOX10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 KROX20 1 1 1 1 1 1 1 OCT6 1 1 1 1 1 1 1 1 1 1 1 SOX2 1 1 1 1 1 1 1 1 1 1 c-myc 1 1 1 1 1 1 1 1 1 1 KLF4 1 1 1 1 1 1 1 1 1 OCT3 / 4 1 1 1 1 1 1 1 1 1 1

[0171] NO. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 evaluate + + + + ++ ++ + ++ + + + + ++ ++ + ++ + + SOX10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 KROX20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OCT6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 SOX2 1 1 1 1 1 1 1 1 1 1 c-myc 1 1 1 1 1 1 1 1 1 1 KLF4 1 1 1 1 1 1 1 1 1 1 OCT3 / 4 1 1 1 1 1 1 1 1 1 1 1

[0172] Example 3 Transformation of normal human skin fibroblasts into Schwann cells and semi-quantification of S100β staining (Fig. 3)

[0173] In with Figure 2 In the same experiment, in order to confirm the staining properties of other combinations, the plate was observed with a fluorescence microscope (manufactured by Olympus) and the staining properties of S100β were evaluated in four stages (+++, ++, +, - in descending order from the substances with more S100β-positive cells).

[0174] use Figure 3 The typical images of each evaluation are shown. In addition, the evaluation results are shown in Table 1. For example, it can be seen that the cells infected with the retroviral vector containing two genes of SOX10 and KROX20 in No. 4 are evaluated as +++, containing the most S100β-positive cells. Even if SOX10 alone or KROX20 alone is introduced, Schwann cells can be induced with low efficiency, but the co-introduction of SOX10 and KROX20 can induce more efficiently. In addition, it can be seen that Oct6 has almost no effect on the induction of Schwann cells.

[0175] Example 4 Transformation from human normal skin fibroblasts to Schwann cells and evaluation of cell morphology (Figure 4)

[0176] In Example 1, the cell morphological changes during the transformation from normal human skin fibroblasts to Schwann cells in cells into which genes for two factors, Sox10 and Krox20, were introduced were evaluated by evaluating the increase in bipolar cell length characteristic of Schwann cells using the cell length / cell width ratio.

[0177] The evaluation results are shown in Fig. 4. The conversion showed an increase in the ratio of cell length to cell width, that is, a change to the characteristic cell morphology of Schwann cells.

[0178] Example 5 Conversion of normal human skin fibroblasts into Schwann cells using other Schwann cells Fluorescence staining of immune cells with associated markers (Figure 5)

[0179] Human normal skin fibroblasts aHDF were cultured on a 12-well plate and the procedure of Example 1 was performed. The results of the group into which two genes, SOX10 and Krox20, were introduced are shown. Immunostaining was performed 12 days after the gene introduction.

[0180] The results are shown in Figure 5. The hosts of anti-p75NTR antibody (primary antibody), anti-GFAP antibody (primary antibody), and anti-NG2 antibody (primary antibody) were rabbits, and the host of anti-Nestin antibody (primary antibody) was mouse. Secondary antibodies used AlexaFluor488,566-labeled anti-rabbit IgG antibody or AlexaFluor488-labeled anti-mouse IgG antibody. Although the positive rate was lower than that of S100β, the expression of each was confirmed.

[0181] Example 6

[0182] A. Transformation of normal human skin fibroblasts into Schwann cells and determination of mRNA expression of S100β and p75NTR genes ( Figure 6 )

[0183] Normal human skin fibroblasts (aHDF) were cultured on a 12-well plate and the procedure of Example 1 was performed. The genes of two factors, Sox10 and Krox20, were introduced into the cells in wells No. 1 to 6, and no genes were introduced into the cells in wells No. 7 to 12 as controls. 12 days after gene introduction, total RNA was recovered from each well using ISOGEN II, and cDNA was synthesized using ReverTra Ace qPCR RTMaster Mix. Real-time PCRMaster Mix, Taqman probe, Specific Primer and cDNA were mixed for the purpose of quantifying the mRNA levels of UCP1 and β-actin genes, and Real-time RT-PCR was performed using AB7300 Real-timePCR system. The mRNA level of the target gene relative to the β-actin mRNA level of each cell was calculated.

[0184] The results are shown in Figure 6 The vertical axis of the graph takes the value of well No. 1 as "1" and represents the relative value of the values ​​of each well as the relative mRNA level. Compared with the control (No. 7 to 12), the cells into which the two genes SOX10 and KROX20 were introduced (No. 1 to 6) showed strong expression of S100β and p75NTR genes, which are Schwann cell-specific markers.

[0185] B. Electrophoresis of RT-PCR products using agarose gel

[0186] The real-time RT-PCR product obtained in A was subjected to electrophoresis using agarose gel. As a result, strong expression of mRNA of S100β and p75NTR gene was confirmed in the cell group into which the genes of two factors, SOX10 and Krox20, were introduced.

[0187] C. The efficiency of direct reprogramming of human fibroblasts into Schwann cells was studied using RT-PCR by the introduction of no factor, SOX10 alone, Krox20 alone, and the combination of SOX10+Krox20.

[0188] aHDF, which is a normal human skin fibroblast, was cultured on a 12-well plate and the operation of Example 1 was performed. The expression of mRNA of S100β and p75NTR in cells without gene introduction (control), cells with only SOX10 gene introduction, cells with only Krox20 gene introduction, and cells with SOX10 gene + Krox20 gene introduction was compared in the same manner as in A. The relative value of mRNA was expressed as the relative mRNA level. The value of the mRNA level of the target gene relative to the mRNA level of β-actin was calculated for each group. The value of one group in the non-introduced cells was taken as "1", and the average of the relative values ​​of the values ​​of each group was expressed as the relative mRNA level. As a result, the expression levels of mRNA of S100β and p75NTR were greatly increased in the group with the two factors of SOX10 + Krox20 introduced. In addition, although the expression levels of mRNA of S100β and p75NTR were increased in the cells with only Sox10 introduction, they were lower than those of the cells with both Sox10 and Krox20 introduction. In addition, even though the mRNA expression level of S100β increased when compared to the cells introduced with both Sox10 and Krox20, the mRNA expression level of S100β was low in the cells introduced with only Krox20. The relative mRNA level of S100β was 4.4 in the non-introduced cells, 1712.3 in the cells introduced with Sox10 alone, 25.1 in the cells introduced with Krox20 alone, and 127615.0 in the cells introduced with Sox10+Krox20. The relative mRNA level of p75NTR was 3.1 in the non-introduced cells, 74.3 in the cells introduced with Sox10 alone, 0.4 in the cells introduced with Krox20 alone, and 35397.6 in the cells introduced with Sox10+Krox20. Therefore, even if the efficiency is low with SOX10 alone or Krox20 alone, direct reprogramming from human fibroblasts to Schwann cells may occur.

[0189] The above results confirm that direct reprogramming from human fibroblasts to Schwann cells is highly efficient when two factors, SOX10 and Krox20, are used. In addition, it is shown that direct reprogramming from human fibroblasts to Schwann cells is possible even with low efficiency using either factor, SOX10 or Krox20.

[0190] Example 7 Study on the effect of dSC on the elongation of neurites of nerve cells (Figure 7)

[0191] NG108-15 was cultured for 12 hours, and then co-cultured with HDF, cSC, or dSC (dSC induced by introducing genes of two factors Sox10 and Krox20) for 36 hours. As a control, NG108-15 was cultured in DMEM supplemented with only 1% FBS. I. The percentage of cells with neurites (neurite-bearing cells), II. The number of primary neurites directly extending from the cell body, and III. The length of the longest neurite (Longest neurite length) were compared and studied by the method of the literature: Tomita K, Madura T, Sakai Y, et al: Glial differentiation of human adipose-derived stem cells: implications for cell-based transplantation therapy. Neuroscience. 2013; 236: 55-65. Only NG108-15 was fluorescently stained using anti-Tuj-1 antibody (primary antibody) and AlexaFluor566-labeled anti-rabbit IgG antibody as secondary antibody.

[0192] The results are shown in Figure 7. By comparison with the control group, both cSC and dSC showed high effects on promoting neurite outgrowth in all parameters studied (I-III), and the performance of dSC was comparable to that of cSC. From the above, it was confirmed that the dSC culture supernatant promoted neurite outgrowth in neurons.

[0193] Example 8 Plasmid introduction (electroporation) from normal human skin fibroblasts induced by non-viral means Transformation to Schwann cells Figure 8 )

[0194] Human normal skin fibroblasts aHDF were cultured on a 12-well plate and the procedure of Example 1 was followed. The results of the group into which two genes, SOX10 and Krox20, were introduced are shown. Immunostaining was performed 14 days after gene introduction.

[0195] The results are shown in Figure 8The host of anti-S100β antibody (primary antibody), anti-p75NTR antibody (primary antibody), and anti-GAP43 antibody (primary antibody) was rabbit, and the secondary antibody was anti-rabbit IgG antibody labeled with AlexaFluor566. Although the positive rate was lower than that in the case of using a retroviral vector, the expression of S100β, p75NTR, and GAP43 was confirmed, and the conversion of fibroblasts aHDF to Schwann cells was confirmed.

[0196] Example 9 Conversion of human normal fat-derived stem cells into Schwann cells and the use of other Schwann cells Immunocytological fluorescence staining images of cell-associated markers (Figure 9)

[0197] The conversion of adipose-derived stromal cells into dSCs was verified.

[0198] <method>

[0199] Human normal fat-derived stromal cells (ADSC: adipose rerived stromal cells) were cultured on a 12-well plate and the procedure of Example 1 was performed. The results of the group into which two genes, SOX10 and Krox20, were introduced are shown. Immunostaining was performed 14 days after the gene introduction.

[0200] <result>

[0201] The results are shown in Figure 9. The host of anti-S100β antibody (primary antibody), anti-p75NTR antibody (primary antibody), anti-GAP43 antibody (primary antibody), and anti-Protein Zero antibody (primary antibody) was rabbit, and the secondary antibody used was anti-rabbit IgG antibody labeled with AlexaFluor566. In the staining with S100β, about 40% of the cells showed positive. Regarding other Schwann cell markers, although the positive rate was lower than that of S100β, their expression could also be confirmed. Protein Zero, a myelin marker, also showed positive.

[0202] Specifically, the induction of Schwann cells by the same method as fibroblasts showed that the cell morphology changed from the morphology of interstitial cells derived from normal human fat to a bipolar or multipolar cell morphology with a relatively small nucleus typical of Schwann cells. Furthermore, among Schwann cells, cells that changed to a typical cell morphology were positive for the Schwann cell marker (S100β) (about 40-50%) ( Fig. 9A ).

[0203] In addition, the induced cells were positive for Schwann cell markers (S100β, GAP43), undifferentiated Schwann cell markers (p75NTR), and myelin markers (P0). Fig. 9B ).

[0204] Example 10 Conversion of umbilical cord vascular endothelial cells to Schwann cells and use of other Schwann cells Immunocytofluorescence staining images of markers (Figure 10)

[0205] The conversion from endothelial cells to dSCs was verified.

[0206] <method>

[0207] Human umbilical vascular endothelial cells (Huvec) were cultured on a 12-well plate and the procedure of Example 1 was performed. The results of the group into which two genes, SOX10 and Krox20, were introduced are shown. Immunostaining was performed 14 days after the gene introduction.

[0208] <result>

[0209] The results are shown in Figure 10. The host of anti-S100β antibody (primary antibody), anti-p75NTR antibody (primary antibody), anti-GAP43 antibody (primary antibody), and anti-Protein Zero antibody (primary antibody) was rabbit, and 566-labeled anti-rabbit IgG antibody was used as the secondary antibody. In the staining with S100β, about 30% of the cells showed positive results. Regarding other Schwann cell markers, although the positive rate was lower than that of S100β, their expression could also be confirmed. Protein Zero, a myelin marker, also showed positive results.

[0210] Specifically, the induction of Schwann cells by the same method as fibroblasts resulted in a change in the cell morphology from that of umbilical cord vascular endothelial cells to a bipolar or multipolar cell morphology with a relatively small nucleus typical of Schwann cells. Furthermore, cells that changed to a typical cell morphology among Schwann cells were positive for the Schwann cell marker (S100β) (about 50-60%) ( Fig. 10A ).

[0211] In addition, the induced cells were positive for Schwann cell markers (S100β, GAP43) and undifferentiated Schwann cell markers (p75NTR) ( Fig. 10B ).

[0212] Example 11 dSCs labeled with GFP (dSCs induced by introduction of genes for two factors, Sox10 and Krox20) and Co-culture of DRGn induces myelination of dSCs in vitro. Immunofluorescence with Schwann cell-associated markers Cell fluorescence staining image (Figure 11)

[0213] The in vitro myelination capacity of dSCs induced by co-culture of GFP-labeled dSCs and DRGn was evaluated.

[0214] <method>

[0215] dSCs were labeled with GFP using a retroviral vector.

[0216] The specific method is based on the method described in the literature: Yoshioka T, Ageyama N, Shibata H, Yasu T, Misawa Y, Takeuchi K, Matsui K, Yamamoto K, Terao K, Shimada K, Ikeda U, Ozawa K, Hanazono Y. Repair of infarcted myocardium mediated by transplanted bone marrow-derived CD34+ stem cells in anonhuman primate model. Stem Cells. 2005 Mar; 23(3): 355-64. and the literature: Hirschmann F, Verhoeyen E, Wirth D, Bauwens S, Hauser H, Rudert M. Vitalmarking of articular chondrocytes by retroviral infection using green fluorescence protein. Osteoarthritis Cartilage. 2002 Feb; 10(2): 109-18.

[0217] Dorsal root ganglion neurons (DRGn) collected from postnatal day 5 mice were cultured in 12-well plates for 2 days and co-cultured in a myelin differentiation induction medium containing GFP-labeled dSCs, nerve growth factor, ascorbic acid, and cAMP. Immunostaining was performed 14 days after the start of co-culture.

[0218] The composition of the culture medium used is as follows:

[0219] DMEM containing N2 supplement (Invitrogen), 50ng / ml ascorbic acid (Wako, Osaka, Japan), and 50ng / ml recombinant rat b-nerve growth factor (NGF) (R&DSystems, Inc., Minneapolis, MN, USA), 0.5μM cAMP (R&DSystems, Inc., Minneapolis, MN, USA).

[0220] The culture medium was prepared according to the literature: Sango K, Kawakami E, Yanagisawa H, Takaku S, Tsukamoto M, Utsunomiya K, Watabe K. Myelination in coculture of established neuronal and Schwann cell lines. Histochem Cell Biol. 2012 Jun; 137(6): 829-39.

[0221] The host of anti-neurofilament antibody (primary antibody) and anti-MBP (Myelin Basic Protein) antibody (primary antibody) is mouse, and the host of anti-Tuj-1 antibody (primary antibody) and anti-Protein Zero antibody (primary antibody) is rabbit. The secondary antibodies are AlexaFluor566-labeled anti-mouse and rabbit IgG antibodies and Cy5-labeled anti-mouse and rabbit IgG antibodies. Anti-MBP antibody and anti-Protein Zero antibody, which are myelin marker antibodies, are labeled in red, and anti-neurofilament antibody and anti-Tuj-1 antibody, which are nerve axon markers, are labeled in gray.

[0222] <result>

[0223] The results are shown in Fig. 11. Myelinated Schwann cells were observed along the neurites of DRG, but some of the myelinated Schwann cells overlapped with the dSC cells labeled with GFP.

[0224] Example 12: GFP-labeled dSCs (2-factor Sox10 and Krox20 introduced) in the sciatic nerve injury model In vitro myelination was induced by transplantation of dSCs induced by genes of the gene promoter. Fluorescence staining of immune cells (Figure 12)

[0225] The myelination ability of dSCs in vivo was investigated.

[0226] <method>

[0227] dSCs were labeled with GFP using a viral vector.

[0228] The sciatic nerve of an immunodeficient mouse was exposed, and the central part was grasped with surgical hemostatic forceps for about 1 minute, and the nerve within a range of about 5 mm was crushed. About 50,000 dSCs labeled with GFP were injected into the distal side. One month after the transplantation, the repaired nerves at the crushed site were collected and immunostained. The outline of the method is shown in Figure 12.

[0229] The host of anti-neurofilament antibody (primary antibody) and anti-MBP antibody (primary antibody) is mouse, the host of anti-Tuj-1 antibody (primary antibody) and anti-Protein Zero antibody (primary antibody) is rabbit, and the secondary antibodies are AlexaFluor566-labeled anti-mouse and rabbit IgG antibodies and Cy5-labeled anti-mouse and rabbit IgG antibodies. Anti-MBP antibody and anti-Protein Zero antibody, which are myelin marker antibodies, are marked in red, and anti-neurofilament antibody and anti-Tuj-1 antibody, which are nerve axon markers, are marked in gray.

[0230] <result>

[0231] The results are shown in Figure 12. GFP+ cells were arranged along the regenerating nerves, and these overlapped with myelin marker+ cells ( Fig. 12B and C).

[0232] GFP+ cells that are erratic parasitized in regenerating nerves also express myelin markers ( Fig. 12B and C, arrows).

[0233] Example 13 Transplantation of dSCs in the Sciatic Defect (5 mm) Model of Immunodeficient Mice ( FIG. 13 )

[0234] <method>

[0235] The cultured Schwann cells (SC: comparative control group) were isolated and cultured from the sciatic nerve. These cells were seeded in gelatin tubes in advance, and a gap of about 5 mm was made in the sciatic nerve trunk of the mouse. The obtained mixed tube was transplanted into the nerve defect. The effect of promoting nerve regeneration was evaluated using Sham mice and the tube transplantation group containing only PBS (Cont) as controls ( Fig.13A ).

[0236] Macroscopic images of reconstructed nerves and myelin staining images of lux fast blue in transverse sections of regenerated nerve tissues were shown. Sciatic nerve function was shown 6 weeks (6W) and 12 weeks (12W) after transplantation, and contraction of the innervated muscles was evaluated by wet muscle weight.

[0237] The Sciatic functional Index (SFI) was determined according to the literature: Inserra MM, Bloch DA, Terris DJ. Functional indices for sciatic, peroneal, and posteriortibial nerve lesions in the mouse. Microsurgery. 1998; 18: 119-124.

[0238] Atrophy and fibrosis of the innervated muscles were evaluated according to the literature: Clavijo-Alvarez JA, Nguyen VT, Santiago LY, Doctor JS, Lee WP, Marra KG. Comparison of biodegradable conduits within aged rat sciatic nerve defects. Plast Reconstr Surg. 2007; 119: 1839-1851.

[0239] <result>

[0240] In the macroscopic images of the reconstructed nerves, both the cSC group and the dSC group were superior to the control group, and no significant difference was observed between the two groups ( Fig. 13B ).

[0241] In the myelin staining images of the transverse sections of the regenerated nerve tissue, the dSC group was comparable to the cSC group ( Fig. 13C ).

[0242] In the Sciatic Functional Index (SFI), at 12W, the dSC group showed recovery of sciatic nerve function comparable to that of cSC ( Fig.13D ).

[0243] Regarding muscle atrophy and fibrosis, significant differences were observed in both groups compared with the control group, but no significant differences were observed between cSC and dSC ( Fig.13E ).

[0244] Example 14 Conversion to Schwann cells (Table 2)

[0245] For the combination of genes shown in Table 2, the same experiment as in Example 2 was performed.

[0246] The combination of which gene is introduced into which well number is recorded in Table 2 (in the table, "1" in the column of each gene indicates infection with a retroviral vector containing the gene, and a blank column indicates that the retroviral vector containing the gene is not infected).

[0247] As in Example 3, the plate was observed using a fluorescence microscope (manufactured by Olympus), and the staining of S100β was evaluated in four stages (+++, ++, +, − in descending order from those with more S100β-positive cells).

[0248] The evaluation results are shown in Table 2.

[0249] This result shows that Oct6 is a factor known to perform important functions in the differentiation of Schwann cells, but it can hardly induce direct reprogramming of somatic cells to Schwann cells. In addition, it is known that Oct6 cannot improve the efficiency of direct reprogramming of somatic cells to Schwann cells caused by Sox10 alone, Krox20 alone, or Sox10+Krox20.

[0250]

Table 2-1

[0251] NO. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 evaluate + + - +++ + - ++ + + - + - - - - - + - - Sox10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 krox20 1 1 1 1 Oct6 1 1 1 1 Sox2 1 1 1 1 1 1 c-myc 1 1 1 1 1 1 KLF4 1 1 1 1 1 Oct4 1 1 1 1 1

[0252] NO. 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 evaluate - - - - - - - - - - - - - - +++ +++ ++ ++ Sox10 1 1 1 1 krox20 1 1 1 1 Oct6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sox2 1 1 1 1 1 1 1 1 1 c-myc 1 1 1 1 1 1 1 1 1 KLF4 1 1 1 1 1 1 1 1 1 Oct4 1 1 1 1 1 1 1 1 1

[0253] NO. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 evaluate - - - - - - - - - - - - - - - - - - Sox10 1 1 1 krox20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Oct6 Sox2 1 1 1 1 1 1 1 1 1 1 c-myc 1 1 1 1 1 1 1 1 1 1 KLF4 1 1 1 1 1 1 1 1 1 1 1 Oct4 1 1 1 1 1 1 1 1 1 1 1

[0254]

Table 2-2

[0255] NO. 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 evaluate ++ ++ ++ ++ + ++ ++ ++ +++ ++ ++ + + + + + + + Sox10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 krox20 1 1 1 1 1 1 1 1 1 1 1 Oct6 1 1 1 1 1 1 1 Sox2 1 1 1 1 1 1 1 1 1 c-myc 1 1 1 1 1 1 1 1 1 1 KLF4 1 1 1 1 1 1 1 1 1 1 Oct4 1 1 1 1 1 1 1 1 1

[0256] NO. 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 evaluate + + + + + + + + - - - - - - - - - - - - Sox10 1 1 1 1 1 1 1 1 krox20 1 1 1 1 1 1 1 1 1 1 1 1 Oct6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sox2 1 1 1 1 1 1 1 1 1 1 1 1 c-myc 1 1 1 1 1 1 1 1 1 1 1 KLF4 1 1 1 1 1 1 1 1 1 1 Oct4 1 1 1 1 1 1 1 1 1 1 1

[0257] NO. 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 evaluate - - - ++ ++ + ++ + + + + ++ ++ + ++ + + - Sox10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 krox20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Oct6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Sox2 1 1 1 1 1 1 1 1 1 1 c-myc 1 1 1 1 1 1 1 1 1 1 KLF4 1 1 1 1 1 1 1 1 1 1 1 Oct4 1 1 1 1 1 1 1 1 1 1 1

[0258] Example 15 Production of neurotrophic factors ( Fig.14 )

[0259] HDF, cSC, and dSC were placed in 4×10 4 cells / cm 2 The cells were seeded in a cell culture dish under the conditions of , and after confirming 80% confluence, they were cultured in a culture medium for collecting cell supernatants for 48 hours. Thereafter, the culture supernatants of each group were passed through a 40-μm filter and collected. For brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF), ELISA kits human BDNF, GDNF, and NGF (Promega, Madison, WI) were used to measure the amount of protein contained in the culture supernatants of each cultured cell.

[0260] As a result, compared with the control (HDF), both cSC and dSC produced all of BDNF, GDNF, and NGF more strongly. In addition, even at the highest point, the production of BDNF was similar between cSC and dSC.

Claims

1. Use of the SOX10 gene or its expression product and the KROX20 gene or its expression product for preparing a composition for directly reprogramming somatic cells into Schwann cells in vivo without via pluripotent stem cells, wherein the somatic cells are fibroblasts, vascular endothelial cells or adipose-derived stem cells.

2. The use according to claim 1, wherein The composition is in a form that can be introduced into somatic cells.

3. The use according to claim 1, wherein The composition comprises a vector into which the SOX10 gene and the KROX20 gene are inserted.

4. The use according to any one of claims 1 to 3, wherein The somatic cells are fibroblasts or vascular endothelial cells.

5. The use according to any one of claims 1 to 3, wherein The somatic cells are fibroblasts located in the nerve injury part of the living body.

6. Use of the SOX10 gene or its expression product and the KROX20 gene or its expression product for producing a vector for directly reprogramming somatic cells into Schwann cells in vivo without via pluripotent stem cells, wherein the somatic cells are fibroblasts, vascular endothelial cells or adipose-derived stem cells.

7. Use of SOX10 gene or its expression product and KROX20 gene or its expression product for producing a composition for generating Schwann cells in vivo from somatic cells without via pluripotent stem cells, the somatic cells being fibroblasts, vascular endothelial cells or adipose-derived stem cells.

8. The use according to claim 7, wherein the composition is a medicament for treating a disease based on nerve deficiency, or a disease based on the deficiency, insufficiency or reduced function of Schwann cells. The disease is selected from the group consisting of cerebral infarction, central nervous system defects or damage caused by spinal cord injury, peripheral nerve defects or damage associated with trauma or resection of neuritis or tumors, multiple sclerosis, neuromyelitis optica (Devic syndrome), concentric sclerosis (Balo disease), acute disseminated encephalomyelitis (ADEM), inflammatory extensive sclerosis (Schilder disease), subacute sclerosing panencephalitis (SSPE), progressive multifocal leukoencephalopathy (PML), Guillain-Barré syndrome, Fisher syndrome, chronic inflammatory demyelinating polyradiculoneuritis, and Charcot-Marie-Tooth disease (CMT).

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