Units used to promote angiogenesis

Abstract

[Problem] To provide a safe means for angiogenesis promotion and / or nerve regeneration (for example, a technique for inducing angiogenesis in transplanted cells or a tissue in vivo) in order to meet requirements for the development of the aforesaid means, a material such as a scaffold enabling the post-transplantation engraftment and growth of nerve stem cells (tissue stem cells of undifferentiated nervous system), etc. [Solution] A unit to be used for angiogenesis promotion and / or nerve regeneration, said unit comprising a gel component and a proteoglycan, etc.

Description

Cross Reference

[0001] This application claims priority in Japan based on Japanese Patent Application No. 2020-069686 filed on April 8, 2020, Japanese Patent Application No. 2020-085736 filed on May 15, 2020, Japanese Patent Application No. 2020-144278 filed on August 28, 2020, and Japanese Patent Application No. 2021-013172 filed on January 29, 2021, the entire contents of which are incorporated herein by reference in their entirety. In addition, the entire contents of all patents, patent applications, and literature cited in this application are incorporated herein by reference in their entirety. [Technical Field]

[0002] The present invention relates to a unit for promoting angiogenesis and / or nerve regeneration in humans and the like. [Background technology]

[0003] Angiogenesis is a phenomenon in animals in which vascular endothelial cells migrate and proliferate from existing capillaries, etc., resulting in the formation of a new vascular network through lumen formation. During the wound healing process (inflammatory, proliferative, and maturation phases), new blood vessels are generated toward the injured area to carry the oxygen and energy necessary for wound healing. Therefore, it is thought that promoting angiogenesis can promote wound healing.

[0004] Vascular endothelial growth factor (VEGF), for example, is known to induce angiogenesis. However, VEGF is known to be produced by tumor cells, for example, and to induce pathological angiogenesis (Patent Document 1). Furthermore, there is a technology for producing endothelial cells from human induced pluripotent stem cells (iPS cells) (Patent Document 2), but it is believed that iPS cells may become cancerous if transplanted while retaining undifferentiated cells, and therefore there are safety concerns regarding the use of this technology.

[0005] Furthermore, due to a shortage of donors and ethical issues, transplantation of neural stem cells is also being considered for the treatment of neurological diseases such as Parkinson's disease and spinal cord injury (Non-Patent Document 1, Patent Document 3).

[0006] The discovery of iPS cells has made it possible, for example, to generate iPS cells from a patient's own cells and then derive neural stem cells from those iPS cells, potentially enabling autologous transplantation, in which a patient's own cells can be used for treatment. However, it is believed to take a long time, more than six months, to generate neural stem cells from a patient's own cells via iPS cells. Furthermore, the safety and differentiation potential of iPS cell lines vary significantly, making it even more time-consuming to verify the safety of iPS cells. Furthermore, in the case of spinal cord injury, neural stem cell transplantation is believed to be ineffective only if the patient's symptoms are not stabilized. Therefore, for diseases in which cell transplantation is effective for a limited period of time, it is considered difficult to use a patient's own cells for treatment, even with iPS cell transplantation technology (Non-Patent Document 1). Furthermore, even in the case of neural stem cell transplantation, the engraftment rate of the transplanted cells is thought to be low (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Takeshi Matsui et al.: STEM CELLS 2012,30:1109-1119 [Non-patent document 2] Koichi Iwatsuki:Spinal Surgery 2015,29:26-31 [Patent documents]

[0008] [Patent Document 1] Patent Publication No. 2016-216375 [Patent Document 2] WO2014 / 192925 [Patent Document 3] Patent Publication No. 2005-278641 Summary of the Invention [Problem to be solved by the invention]

[0009] Currently, there is a need for safe means for promoting angiogenesis and / or nerve regeneration (for example, techniques for inducing angiogenesis in cells or tissues transplanted into the body), as well as the development of agents such as scaffolds that allow neural stem cells (undifferentiated tissue stem cells of the nervous system) to take root and proliferate after transplantation.

[0010] Therefore, an object of the present invention is to provide a means for safely promoting angiogenesis. [Means for solving the problem]

[0011] The present invention has been made to solve such problems, and one aspect of the present invention is "a unit used for promoting angiogenesis and / or nerve regeneration, comprising a gel component and a proteoglycan," and the present invention is, for example, the following inventions (1) to (11).

[0012] (1) A unit used for promoting angiogenesis and / or nerve regeneration, comprising a gel component and a proteoglycan. (2) The unit according to (1), which contains VEGF. (3) The unit according to (1) or (2), which contains FGF-2. (4) A unit according to any one of (1) to (3), which contains EGF. (5) A unit described in any one of (1) to (4), further comprising cells for promoting angiogenesis. (6) A unit according to any one of (2) to (5), in which a liquid medium containing VEGF and cells for promoting angiogenesis are layered on top of the gel component. (7) A unit described in any one of (1) to (6), having a proteoglycan content of 0.1 μg / ml or more. (8) The unit according to any one of (2) to (7), wherein the proteoglycan is contained in a liquid medium containing VEGF. (9) A unit according to any one of (1) to (8), wherein the proteoglycan is contained in a gel component. (10) The unit according to any one of (1) to (9), wherein the proteoglycan is a chondroitin sulfate proteoglycan. (11) The unit according to (10), wherein the proteoglycan is derived from salmon nasal cartilage. [Effects of the Invention]

[0013] By using this unit, it is possible to safely promote angiogenesis within the body, for example, by implanting the unit into the body of a human. [Brief explanation of the drawings]

[0014] [Figure 1] The results of evaluation of angiogenic potential (photographs taken under a phase-contrast microscope, 4x magnification) are shown in the figure. In the figure, (1) is a photograph of the PG0 group taken 2 hours after the start of culture, (2) is a photograph of the PG1000 group taken 2 hours after the start of culture, (3) is a photograph of the GAG1000 group taken 2 hours after the start of culture, (4) is a photograph of the PG0 group taken 4 hours after the start of culture, (5) is a photograph of the PG1000 group taken 4 hours after the start of culture, (6) is a photograph of the GAG1000 group taken 4 hours after the start of culture, (7) is a photograph of the PG0 group taken 6 hours after the start of culture, (8) is a photograph of the PG1000 group taken 6 hours after the start of culture, and (9) is a photograph of the GAG1000 group taken 6 hours after the start of culture. [Figure 2]The results of measuring blood vessel length (Figure (a)), number of meshwork structures (Figure (b)), and number of branching points (Figure (c)) in samples from each group shown in Figure 1 taken 6 hours after the start of culture were obtained. The results were obtained by quantifying each item (blood vessel length, number of meshwork structures, and number of branching points) in four randomly selected fields of view, and calculating the average of the quantified values. Statistical analysis was performed using Dunnett's method, with * indicating a significant difference (p<0.05). The measurements were performed by culturing the blood vessels three times and calculating the average of the three measurements. The blood vessel length (pixels) for each group was 3187 in the PG0 group, 3192 in the PG1000 group, and 2996 in the GAG1000 group (significantly different from the PG0 group). The number of mesh structures in each group was 9.9 in the PG0 group, 10.9 in the PG1000 group (significantly different from the PG0 group), and 8.2 in the GAG1000 group (significantly different from the PG0 group).The number of branch points in each group was 16.7 in the PG0 group, 20.4 in the PG1000 group (significantly different from the PG0 group), and 14.6 in the GAG1000 group (significantly different from the PG0 group). [Figure 3] The results of evaluation of angiogenic activity are shown (photographs taken by observation using a phase contrast microscope, magnification: 4x). In the figure, (1) is a photograph of the PG0 group taken 2 hours after the start of culture, (2) is a photograph of the PG1 group taken 2 hours after the start of culture, (3) is a photograph of the PG10 group taken 2 hours after the start of culture, (4) is a photograph of the PG100 group taken 2 hours after the start of culture, (5) is a photograph of the PG1000 group taken 2 hours after the start of culture, (6) is a photograph of the PG0 group taken 4 hours after the start of culture, (7) is a photograph of the PG1 group taken 4 hours after the start of culture, (8) is a photograph of the PG10 group taken 4 hours after the start of culture, (9) is a photograph of the PG100 group taken 4 hours after the start of culture, (10) is a photograph of the PG1000 group taken 4 hours after the start of culture, (11) is a photograph of the PG0 group taken 6 hours after the start of culture, (12) is a photograph of the PG1 group taken 6 hours after the start of culture, (13) is a photograph of the PG10 group taken 6 hours after the start of culture, (14) is a photograph of the PG100 group taken 6 hours after the start of culture, and (15) is a photograph of the PG1000 group taken 6 hours after the start of culture. [Figure 4]The results of measuring blood vessel length (Figure (a)), number of meshwork structures (Figure (b)), and number of branching points (Figure (c)) in samples from each group shown in Figure 3 6 hours after the start of culture were obtained. The culture was performed three times and the average of the three measurements was calculated. The results were obtained by quantifying each item (blood vessel length, number of meshwork structures, and number of branching points) in four randomly selected fields and calculating the average of the quantified values. The measurements are shown as relative values ​​with PG0 set to 100. Statistical analysis was performed using Dunnett's method, and * indicates a significant difference: p<0.05 (indicated as * in the figure), p<0.01 (indicated as ** in the figure), p<0.001 (indicated as *** in the figure), and p<0.0001 (indicated as **** in the figure). The blood vessel length for each group was 108.20 for the PG1 group, 120.87 for the PG10 group (significantly different from the PG0 group), 129.44 for the PG100 group (significantly different from the PG0 group), and 123.17 for the PG1000 group, with the PG0 group set to 100. The number of reticular structures for each group was 125.23 for the PG1 group (significantly different from the PG0 group), 148.17 for the PG10 group (significantly different from the PG0 group), 170.85 for the PG1000 group (significantly different from the PG0 group), and 159.66 for the PG1000 group, with the PG0 group set to 100. The number of branching points for each group, with the PG0 group set at 100, was 127.68 for the PG1 group (significantly different from the PG0 group), 149.59 for PG10 (significantly different from the PG0 group), 166.24 for PG100 (significantly different from the PG0 group), and 161.37 for PG1000 (significantly different from the PG0 group). [Figure 5] The results of evaluating angiogenic potential (photographs taken under a phase-contrast microscope at 4x magnification) are shown in the figure. (1) is a photograph of the PG0 group taken 2 hours after the start of culture, (2) is a photograph of the PG100 group taken 2 hours after the start of culture, (3) is a photograph of the PG0 group taken 4 hours after the start of culture, (4) is a photograph of the PG100 group taken 4 hours after the start of culture, (5) is a photograph of the PG0 group taken 6 hours after the start of culture, and (6) is a photograph of the PG100 group taken 6 hours after the start of culture. [Figure 6]The results of measuring blood vessel length (Figure (a)), number of meshwork structures (Figure (b)), and number of branching points (Figure (c)) in samples from each group shown in Figure 5 6 hours after the start of culture were obtained. The results were obtained by quantifying each item (blood vessel length, number of meshwork structures, and number of branching points) in four randomly selected fields of view and calculating the average of the quantified values. Statistical analysis was performed using Dunnett's method, with * indicating a significant difference, p<0.05 (indicated as * in the figure) and p<0.01 (indicated as ** in the figure). The measurements were performed by performing the culture twice and calculating the average of the two measurements. The blood vessel length (pixels) in each group was 2702 in the PG0 group and 3368 in the PG1000 group (significantly different from the PG0 group). The number of mesh structures in each group was 6.49 in the PG0 group and 9.75 in the PG100 group (significantly different from the PG0 group). The number of branch points in each group was 12.5 in the PG0 group and 17.8 in the PG100 group (significantly different from the PG0 group). [Figure 7] Results of Experiment 3 (confirmation of proteoglycan localization by fluorescence microscopy): (1) shows the results of staining with DAPI for the labeled PG in medium (without Matrigel) group, (2) shows the results of staining with labeled PG1 for the labeled PG in medium (without Matrigel) group, (3) shows the results of staining with Merge for the labeled PG in medium (without Matrigel) group, (4) shows the results of staining with DAPI for the labeled PG in medium group, (5) shows the results of staining with labeled PG1 for the labeled PG in medium group, (6) shows the results of staining with Merge for the labeled PG in medium group, (7) shows the results of staining with DAPI for the labeled PG in gel group, (8) shows the results of staining with labeled PG1 for the labeled PG in gel group, and (9) shows the results of staining with Merge for the labeled PG in gel group. "DAPI" indicates that the nuclei of HUVECs were labeled by DAPI staining, "Fluorescein" indicates that the proteoglycans were labeled with labeled PG1, and "Merge" indicates that the nuclei and proteoglycans were labeled together. [Figure 8] This shows the results of Experiment 3 (confirmation of the localization of proteoglycans using a confocal microscope), and is a diagram of (4), (6), (7), and (9) extracted from Figure 7. [Figure 9]Results of Experiment 4 (confirmation of proteoglycan localization using a confocal microscope). "Hoechst" indicates that the nuclei of HUVECs were labeled using Hoechst staining, "Cell mask" indicates that the cell membrane of HUVECs was labeled using CellMask™ staining, "PG-Atto590" indicates that the proteoglycans were labeled using labeled PG2, and "Merge" indicates that the nuclei, cell membrane, and proteoglycans were labeled together. (1) is the result of observation using the microscope at 20x magnification for "Hoechst," (2) is the result of observation using the microscope at 20x magnification for "Cell mask," (3) is the result of observation using the microscope at 20x magnification for "PG-Atto590," (4) is the result of observation using the microscope at 20x magnification for "Merge," (5) is the result of observation using the microscope at 40x magnification for "Hoechst," and (6) is the result of observation using the microscope at 40x magnification for "Cell mask." (7) is the result of observation of "PG-Atto590" under the microscope at 40x magnification, (8) is the result of observation of "Merge" under the microscope at 40x magnification, and (9) is the result of three-dimensional observation of "Merge" under the microscope at 60x magnification.

[0015] [Figure 10] These are the results of evaluating angiogenic activity in Experiment 5 (photographs taken under a phase-contrast microscope at 4x magnification). In the figure, (1) is a photograph of the PG0 group taken 6 hours after the start of culture, (2) is a photograph of the PG100 group taken 6 hours after the start of culture, and (3) is a photograph of the bovine PG100 group taken 6 hours after the start of culture. [Figure 11] These are the results of evaluating angiogenic potential using an aortic ring assay (results of Experiment 6). [Figure 12] These are the results of evaluating the regenerative ability of nerves and / or blood vessels using a sciatic nerve injury model (results of Experiment 7). [Figure 13] These are the results of evaluating angiogenic activity using a sciatic nerve injury model (results of Experiment 8). [Figure 14]The results of experiment 9 show the results of evaluating angiogenesis and other properties using a sciatic nerve injury model. The bar in Fig. 14 indicates 500 μm. The dotted line in Fig. 14 indicates the area of ​​the sciatic nerve that was injured when the model mouse was created.

[0016] [Figure 15] These are the results of evaluating angiogenic activity using a sciatic nerve injury model (results of Experiment 11). [Figure 16] These are the results of Experiment 12. [Figure 17] This is the result of Experiment 12. The bar shown in this Figure 17 indicates 20 μm. [Figure 18] This is the result of Experiment 12. The bar shown in this Figure 18 indicates 200 μm.

[0017] [Figure 19] FIG. 1 is a schematic diagram of the experimental procedure performed in Experiment 13. [Figure 20] This shows the results of measuring the number of neurospheres formed in each treatment group. The vertical axis indicates the percentage of neurospheres (NS) formed, specifically the percentage of NS formed (%) when the number of NS formed in the control group was set at 100 (%). Statistical analysis was performed using Dunnett's method, and * indicates a significant difference (p<0.05). [Figure 21] This shows the results of observation of neurospheres in each administration group. [Figure 22] FIG. 1 is a schematic diagram of the experimental procedure performed in Experiment 14. [Figure 23] This is a schematic diagram of the experimental procedure performed in Experiment 15. [Figure 24] This is a schematic diagram of the experimental procedure performed in Experiment 16. [Figure 25]These are the results of Experiment 16 (confirmation of the localization of proteoglycans in neural stem cells). The "medium 2 group" is the group cultured under medium 2 conditions (group cultured in medium containing unlabeled proteoglycans), and the "medium 8 group" is the group cultured under medium 8 conditions (group cultured in medium containing labeled proteoglycans). "Hoechst" indicates that the nuclei of neural stem cells were labeled using Hoechst staining, and "Fluorescein-PG" indicates that the localized proteoglycans were labeled using fluorescent staining. [Figure 26] These are the results of Experiment 17 (Confirmation of the localization of proteoglycans in neural stem cells (confirmation using a confocal microscope)). "Hoechst" indicates that the nuclei of neural stem cells were labeled with Hoechst staining, "Cell mask" indicates that the cell membrane of neural stem cells was labeled, "PG-Atto590" indicates that proteoglycans were labeled, and "Merge" indicates that the nucleus, cell membrane, and proteoglycan labels were combined. DETAILED DESCRIPTION OF THE INVENTION

[0018] The unit according to the present invention will be described below.

[0019] (VEGF) Vascular endothelial growth factor (VEGF) is a growth factor that specifically acts on vascular endothelial cells isolated from cultured pituitary foliculo-stellate cells. VEGF is a member of the cysteine-knot growth factor superfamily and plays an important role in de novo angiogenesis (vasculogenesis) during the fetal stage and in angiogenesis (the formation of new blood vessels by branching and elongating pre-existing vessels). VEGF is a member of the VEGF family of proteins, consisting of VEGF-A and VEGF-B. VEGF-A promotes endothelial cell proliferation, vascular permeability, and duct formation, and induces the production of active substances from endothelial cells. VEGF-E specifically promotes angiogenesis in tumor tissue.

[0020] (liquid medium) The medium used in the present invention is preferably a liquid medium, from the viewpoint of ease of medium preparation (for example, it is thought that medium preparation is easier when performing suspension culture). Known media can be used as the liquid medium. Examples include media containing components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, vitamins) (e.g., Iscove's Modified Dulbecco's Medium (IMDM), RPMI, DMEM (e.g., DMEM / Ham's F-12 medium (Nacalai Tesque, Inc., 08460-95) used in the following Examples), Fischer's medium, α medium, Leibovitz medium, L-15 medium, NCTC medium, F-12 medium, MEM, McCoy's medium), etc. Furthermore, if necessary, further additives (serum, antibiotics, etc.) may also be added.

[0021] (angiogenesis) Angiogenesis is an important morphogenetic process that creates new blood vessel networks in vivo. Vascular endothelial cells cooperate with other cells to promote the formation of new blood vessels in a wound-specific manner. (More specifically, vascular endothelial cells cooperate with other cells to create characteristic vascular structures, such as two- and three-dimensional branched structures, through repeated processes of sprouting, elongation, branching, and lumen formation.) Normal (physiological) angiogenesis is responsible for various biological processes, such as fetal angiogenesis, endometrial formation, follicle formation, and wound healing. Pathological angiogenesis is responsible for various biological processes, such as malignant tumor formation, ocular neovascular disease, rheumatoid arthritis, and atherosclerosis. Insufficient angiogenesis can lead to conditions such as arteriosclerosis obliterans, angina pectoris, and myocardial infarction. To treat diseases caused by insufficient angiogenesis, revascularization therapy, which promotes angiogenesis, is used.

[0022] (Cells that promote angiogenesis) The cells used in the present invention to promote angiogenesis are not particularly limited in type, but are mammalian cells such as human cells, such as somatic cells, precursor cells of these somatic cells, or a mixture thereof. Examples of somatic cells used in the present invention to promote angiogenesis include cardiomyocytes, endothelial cells (vascular endothelial cells, lymphatic endothelial cells, etc.), mural cells (pericytes, etc.), and muscle cells (skeletal muscle cells, smooth muscle cells, etc.). In the following examples, HUVECs (human umbilical vein endothelial cells) were used as cells to promote angiogenesis.

[0023] (Gel component) The gel components (materials) used in the present invention are typically biocompatible, capable of containing large amounts of water, and capable of facilitating the diffusion and transport of substances necessary for cell survival, such as oxygen, water, nutrients, and enzymes. The form of the gel components is not particularly limited as long as they can be incorporated into the unit, and examples include particle, granule, film, tube, disk, net, mesh, porous, suspension, and dispersion forms. Examples of gel components include hydrogels such as gelatin hydrogel. The gel components can also contain various components, such as laminin, type IV collagen, entactin / nidogen, and specific growth factors, as needed to regulate cell proliferation and promote angiogenesis. In the following examples, examples of gel components used include Matrigel (registered trademark, Corning, Product Number: 356231) and Collagen Gel Culturing Kit cell matrix type 1-A (Nitta Gelatin, 638-00781).

[0024] (proteoglycan) Proteoglycans are glycoproteins composed of a core protein covalently bound to sugar chains called glycosaminoglycans (GAGs), such as chondroitin sulfate and dermatan sulfate. Proteoglycans are one of the major components of the extracellular matrix and are widely distributed throughout the body, including in skin and cartilage. GAG chains have a long, unbranched, linear structure. They are negatively charged due to the large number of sulfate and carboxyl groups they contain, and the electrical repulsion between them causes the GAG ​​chains to assume an extended shape. Furthermore, proteoglycans can retain large amounts of water due to the water affinity of sugars. The numerous GAG chains contained in proteoglycans retain water flexibly like a sponge, providing cartilage with its unique functions of elasticity and shock resistance.

[0025] The core protein of proteoglycans has the property of binding to various molecules in the matrix. Cartilage proteoglycans have binding domains at the N-terminus that bind to hyaluronic acid and link proteins, and can bind to these substances or even associate with each other in the same molecule. The C-terminus contains lectin-like domains and EGF-like domains that bind to various other molecules. This property allows proteoglycans to build structures that are suited to each tissue. Among proteoglycans, chondroitin sulfate proteoglycans are proteoglycans in which chondroitin sulfate is covalently bound to a core protein.

[0026] Salmon nasal cartilage-derived proteoglycan is a proteoglycan extracted from salmon nasal cartilage. Here, salmon is, for example, a fish belonging to the genus Oncorhynchus, and preferably salmon with the scientific name "Oncorhynchus keta" is selected from the viewpoint of cell proliferation (e.g., more efficient cultivation of target cells). Furthermore, the lower limit of the content of proteoglycan contained in the unit of the present invention is preferably 0.1 μg / ml or more, more preferably 0.2 μg / ml or more, and even more preferably 0.5 μg / ml or more, from the viewpoint of, for example, cell proliferation. The proteoglycan contained in the unit according to the present invention is prepared, for example, by the method described in the publication (Japanese Patent No. 6317053). In the experiments shown in the following examples, the proteoglycan used was, for example, a proteoglycan derived from salmon nasal cartilage (Fujifilm Wako Pure Chemical Industries, Ltd. (product codes: 162-22131, 168-22133)) and a proteoglycan derived from bovine nasal septum.

[0027] In the unit according to the present invention, the proteoglycan is contained in the liquid medium and / or the gel component.

[0028] (unit) As described above, the unit according to the present invention is a "unit (population) used for promoting angiogenesis, comprising a gel component and a proteoglycan," a "unit (population) comprising a liquid medium containing VEGF, cells for promoting angiogenesis, a gel component, and a proteoglycan," etc. However, it is also possible to further use a gel component to prepare a stack of multiple units, and to transplant the stack into the body of a human or the like.

[0029] The unit of the present invention can be used, for example, in cell therapy such as cell transplantation (a treatment method that uses one's own cells or cells from another person to treat a disease), and regeneration-inducing medicine (a treatment that locally mobilizes stem cells present in the body to tissue damaged by injury or disease without removing them from the body, thereby inducing functional tissue regeneration).

[0030] (FGF-2) FGF-2 (Fibroblast Growth Factor-2), also known as bFGF, can be isolated from animal nervous tissue, pituitary gland, adrenal cortex, placenta, etc. FGF-2 is known to induce neural differentiation, survival, and regeneration, as well as regulate embryonic development and differentiation. FGF-2 has a wide range of functions, including as a cell growth factor, angiogenic factor, and neurotrophic factor, and exhibits proliferative activity in various cells, including ES cells and iPS cells.

[0031] (EGF) EGF (Epidermal Growth Factor) is a polypeptide that promotes the proliferation of various cells, including epithelial cells. The action of EGF is not species-specific, and it exhibits a proliferation effect on a variety of cells, including epithelial cells, fibroblasts, and hepatocytes. It is thought to be a growth factor that plays an important role in the proliferation and differentiation of cells in the body.

[0032] (neural stem cells) Neural stem cells are undifferentiated tissue stem cells of the nervous system that possess both self-renewal and pluripotency. In the embryonic brain of mammals such as humans, neural stem cells first exponentially increase their numbers by actively proliferating, and then undergo asymmetric division. Furthermore, in the late embryonic and postnatal brain, neural stem cells are known to give rise to astrocytes and oligodendrocytes. Neural stem cells are the source of neurons, astrocytes, and oligodendrocytes, the major cell types that make up the central nervous system. In the present invention, cells derived from mammals such as humans and mice are preferably used.

[0033] (Neural regeneration) Nerve regeneration, such as of central and / or peripheral nerves, at least partially reproduces the normal developmental process in nerves, regardless of the origin of the regenerating cells. Examples of regenerating cells include stem cells (e.g., neural stem cells, embryonic stem cells, bone marrow cells, etc.), neural progenitor cells, or neurons. Furthermore, cells used for nerve regeneration may be endogenous cells (e.g., neural stem cells, neural progenitor cells, neurons, mature neurons, etc.) or exogenous cells (e.g., transplanted neural stem cells, transplanted neural progenitor cells, transplanted neurons, transplanted mature neurons, etc.). Exogenous cells may be autologous or allogeneic. Nerve regeneration encompasses tissue regeneration or functional regeneration, including, for example, the engraftment, differentiation, proliferation, and / or maturation of the aforementioned cells. Maturation refers, for example, to the growth of neurons into a functional state capable of signal transduction, etc. Regeneration also encompasses, for example, neurotrophic factor-like effects and neurotrophic factor activity enhancement. For example, peripheral nerve regeneration involves the extension of peripheral nerves damaged by external or internal factors to target cells, the reconstruction of neural circuits, and the prevention and / or treatment of peripheral nerve disorders. [Example]

[0034] [Experiment 1: Evaluation of angiogenic potential] The angiogenic potential of units containing proteoglycans and the like was evaluated using human umbilical vein endothelial cells (hereinafter referred to as "HUVECs") derived from normal human newborns.

[0035] The experimental method is described below. First, Matrigel (150 μl / well) was placed in each well and left to stand for 10 minutes at room temperature (23°C). After this standing, the wells were further left to stand for 30 minutes at 37°C in a 5% CO2 environment. After this 30-minute standing, culture medium (EGM) was added to each well. TM -2 BulletKit TM ) was added, and equilibration was carried out for 15 minutes at 37°C in a 5% CO environment. After the equilibration, HUVECs (5.0 × 10 4 Cells were seeded at 1000 x 1000 cells / well and cultured at 37°C in 5% CO2 in the medium for each of the following groups (PG0, PG1, PG10, PG100, PG1000, GAG1000). Evaluations were performed 2 hours (indicated as 2h in the figure), 4 hours (indicated as 4h in the figure), and 6 hours (indicated as 6h in the figure) after the start of culture.

[0036] In this experiment 1, the composition of the medium for each group (PG0, PG1, PG10, PG100, PG1000, GAG1000) is as follows.

[0037] (PG0) Culture medium (EGM TM -2 BulletKit TM ) does not contain proteoglycans.

[0038] (PG1) Culture medium (EGM TM -2 BulletKit TM ) medium to which proteoglycan was added to a final concentration of 1 μg / ml.

[0039] (PG10) Culture medium (EGM TM -2 BulletKit TM) medium to which proteoglycan was added to a final concentration of 10 μg / ml.

[0040] (PG100) Culture medium (EGM TM -2 BulletKit TM ) to which proteoglycan was added to a final concentration of 100 μg / ml.

[0041] (PG1000) Culture medium (EGM TM -2 BulletKit TM ) to which proteoglycan was added to a final concentration of 1000 μg / ml.

[0042] (GAG1000) Culture medium (EGM TM -2 BulletKit TM ) to which GAG was added to a final concentration of 1000 μg / ml.

[0043] The results of Experiment 1 are shown in Figures 1 to 4. The experimental results shown in Figures 1 and 2 indicate that angiogenesis is promoted by adding proteoglycan to the culture medium, but that angiogenesis is not promoted when GAG (which, unlike proteoglycan, has no core protein bound to it) is added to the culture medium. The experimental results shown in Figures 3 and 4 indicate that angiogenesis is promoted by adding proteoglycan to the culture medium in a concentration-dependent manner (at least up to the group to which 100 μg / ml of proteoglycan was added).

[0044] [Experiment 2: Evaluation of angiogenic potential] Using HUVECs, we evaluated the angiogenic potential of units containing proteoglycans, etc. Unlike Experiment 1, in Experiment 2, we evaluated the angiogenic potential of Matrigel by adding a predetermined amount of proteoglycan.

[0045] The experimental method is described below. Matrigel (150 μl / well, no proteoglycan added) or Matrigel (150 μl / well) containing 100 μg / ml of proteoglycan added was added to each well and allowed to stand at room temperature (23°C) for 10 minutes. In Figures 5 and 6, "PG0" represents the Matrigel (150 μl / well, no proteoglycan added) group, and "PG100" represents the Matrigel (150 μl / well) containing 100 μg / ml of proteoglycan added group. After this standing, the plates were further left standing for 30 minutes in an environment of 37°C and 5% CO2. After this 30-minute standing, a culture medium (EGM TM -2 BulletKit TM ) was added to each well, and equilibration was carried out at 37°C in a 5% CO environment for 15 minutes. After equilibration, HUVECs (5.0 × 10 4 Cells (cells / well) were seeded and cultured at 37°C in a 5% CO environment. Predetermined evaluations were performed 2 hours (indicated as 2h in the figure), 4 hours (indicated as 4h in the figure), and 6 hours (indicated as 6h in the figure) after the start of culture.

[0046] The results of Experiment 2 are shown in Figures 5 and 6. The experimental results shown in Figures 5 and 6 demonstrated that angiogenesis was promoted even when proteoglycan was added to Matrigel rather than to the culture medium.

[0047] [Experiment 3: Confirmation of proteoglycan localization during angiogenesis] Using HUVECs, the localization of proteoglycans in angiogenesis was confirmed using fluorescence and confocal microscopes.

[0048] The procedure for Experiment 3 is described below. First, the following three types of wells are prepared. Group without Matrigel: In Figure 7, this is indicated as "Labeled PG in medium (no Matrigel)." Matrigel-added group (70 μl / well): In Figures 7 and 8, this is referred to as "labeled PG in medium." Matrigel was added (70 μl / well), and "labeled PG1" was added to this Matrigel so that the final concentration in the Matrigel and culture medium unit was 100 μg / ml: this group is shown as "labeled PG in gel" in Figures 7 and 8.

[0049] After preparing these wells, they were left to stand at room temperature (23°C) for 10 minutes, and then further left to stand at 37°C in a 5% CO2 environment for 30 minutes.

[0050] After leaving it for 30 minutes, culture medium (EGM TM -2 BulletKit TM ) was added to each well, and equilibration was carried out at 37°C in a 5% CO environment for 15 minutes. After equilibration, HUVECs (5.0 × 10 4 In the group to which Matrigel was added (70 μl / well) (labeled PG group), "labeled PG1" was further added to the culture medium so that the final concentration in the unit of Matrigel and culture medium was 100 μg / ml.

[0051] After seeding, the cells were cultured for 2 hours at 37°C in an environment of 5% CO2. After the culture, the cells were collected from each well by centrifugation at 400g for 3 minutes. The collected cells were washed with PBS for 5 minutes. After the washing, the cells were fixed with 4% paraformaldehyde for 15 minutes. After the fixation, the cells were washed again with PBS for 5 minutes. After the washing, the cells were transferred onto a slide glass with DAPI (Dojindo Laboratories, D212-Cellstain). TM The specimens were mounted with a mounting medium containing DAPI (DAPI). Using a fluorescence microscope (Olympus Corporation: Inverted Research Microscope IX81) and a confocal microscope (Olympus Corporation: Confocal Laser Scanning Microscope FV3000) at 40x magnification, the localization of HUVEC nuclei (stained with DAPI) and proteoglycans was compared.

[0052] The observation results are shown in Figures 7 and 8. In Figures 7 and 8, blue indicates that the HUVEC nuclei were stained with DAPI, and green indicates that the proteoglycans were labeled. In the group with labeled PG in the medium (without Matrigel), no proteoglycan localization was confirmed (Figure 7 (1) to (3)). On the other hand, in the group with labeled PG in the medium and the group with labeled PG in the gel (Figure 7 (4) to (9) and Figure 8), it was confirmed that already 2 hours after the start of cell culture, there was a high correlation between the localization of endothelial cells and the localization of proteoglycans (specifically, proteoglycans were distributed along the blood vessels that were attempting to form a ring structure).

[0053] [Experiment 4: Confirmation of proteoglycan localization in angiogenesis] The localization of proteoglycans in angiogenesis was confirmed using HUVECs under fluorescent and confocal microscopes. The labeled proteoglycan used in Experiment 4 was labeled PG1, not PG2, as used in Experiment 3.

[0054] The procedure for Experiment 4 is described below. First, the following two types of wells are prepared. Group without Matrigel addition: group not shown in FIG. Matrigel was added (70 μl / well), and proteoglycan was further added to this Matrigel so that the final concentration in the Matrigel and culture medium unit was 100 μg / ml: the group shown in FIG. 9.

[0055] After preparing these wells, they were left to stand at room temperature (23°C) for 10 minutes, and then further left to stand at 37°C in a 5% CO2 environment for 30 minutes.

[0056] After leaving it for 30 minutes, culture medium (EGM TM -2 BulletKit TM ) was added to each well, and equilibration was carried out at 37°C in a 5% CO environment for 15 minutes. After equilibration, HUVECs (5.0 × 10 4In the group to which Matrigel was added (70 μl / well) (labeled PG group), "labeled PG1" was further added to the culture medium so that the final concentration in the unit of Matrigel and culture medium was 100 μg / ml.

[0057] After seeding, the cells were cultured for 2 hours at 37°C in a 5% CO environment. One hour before harvesting the cells (cultured cells) to be used for observation, Hoechst 33342 (final concentration 10 mg / mL, Hoechst 33342) was added to stain the cell nuclei. TM 33342 Imaging Protocol (Thermo Fisher Scientific)) and Cellmask (final concentration 5 mg / mL, CellMask) to stain the cell membrane. TM The cells were stained with a stain (Thermo Fisher Scientific). After the incubation, the cells were collected by centrifugation at 400 g for 3 minutes. The collected cells were washed with PBS for 5 minutes. After the washing, the cells were fixed with 4% paraformaldehyde for 15 minutes. After fixation, the cells were washed again with PBS for 5 minutes. After the washing, the cells were mounted on a glass slide with mounting medium to prepare an observation sample. Using a confocal microscope (Olympus Corporation: Confocal Laser Scanning Microscope FV3000) at 20x, 40x, and 60x magnifications, the localization of HUVEC nuclei (stained with Hoechst as described above), HUVEC cell membranes (stained with Cellmask as described above), and proteoglycans was compared.

[0058] The observation results are shown in Figure 9. In Figure 9, "Hoechst" indicates that the nuclei of HUVECs were labeled with Hoechst staining, and "Cell mask" indicates that the nuclei were labeled with CellMask. TMThe "PG-Atto590" indicates the labeling of the HUVEC cell membrane by staining, "PG-Atto590" indicates the labeling of proteoglycans with PG2, and "Merge" indicates the combined labeling of the nucleus, the cell membrane, and the proteoglycan. The results shown in Figure 9, similar to those shown in Figures 7 and 8, confirmed that even 2 hours after the start of cell culture, there was a high correlation between the localization of endothelial cells and the localization of proteoglycans (specifically, proteoglycans were distributed along the blood vessels attempting to form a ring structure). Although not shown in Figure 9, the localization shown in Figure 9 was not observed in the group without Matrigel.

[0059] [Experiment 5: Evaluation of angiogenic activity] The angiogenic potential of the units containing proteoglycans was evaluated using HUVECs.

[0060] The experimental method is described below. First, Matrigel (150 μl / well) was placed in each well and left to stand for 10 minutes at room temperature (23°C). After this standing, the wells were further left to stand for 30 minutes at 37°C in a 5% CO2 environment. After this 30-minute standing, culture medium (EGM) was added to each well. TM -2 BulletKit TM ) was added, and equilibration was carried out for 15 minutes at 37°C in a 5% CO environment. After the equilibration, HUVECs (5.0 × 10 4 Cells were cultured at 37°C in 5% CO2 under the medium conditions of the following groups (PG0, PG100, bovine PG100). Six hours after the start of culture, the cells were evaluated as specified.

[0061] In this experiment 5, the composition of the medium for each group (PG0, PG100, bovine PG100) was as follows.

[0062] (PG0) Culture medium (EGM TM -2 BulletKit TM ) does not contain proteoglycans.

[0063] (PG100) Culture medium (EGM TM -2 BulletKit TM ) to which proteoglycan (proteoglycan derived from salmon nasal cartilage) was added to a final concentration of 100 μg / ml.

[0064] (Beef PG100) Culture medium (EGM TM -2 BulletKit TM ) to which proteoglycan derived from bovine nasal septum was added to a final concentration of 100 μg / ml.

[0065] The results of Experiment 5 are shown in Figure 10 and Tables 1 to 3 below. Tables 1 to 3 show the results of measuring the blood vessel length (Table 1), number of meshwork structures (Table 2), and number of branching points (Table 3) in samples taken 6 hours after the start of culture for each group shown in Figure 10. The results shown in Tables 1 to 3 were obtained by quantifying each item (blood vessel length, number of meshwork structures, number of branching points) for four randomly selected fields of view and calculating the average of the quantified values. In Tables 1 to 3, the measured values ​​are shown relative to PG0, which is set to 100, and SD is the standard deviation.

[0066] The results of Experiment 5 showed that angiogenesis was promoted not only in the group where proteoglycan derived from salmon nasal cartilage was added to the culture medium (PG100), but also in the group where proteoglycan derived from bovine nasal septum was added (bovine PG100).

[0067] [Table 1]

[0068] [Table 2]

[0069] [Table 3]

[0070] In Experiments 1 to 5, the following samples were used: HUVEC (C2517A and C2517AS, LONZA): Human umbilical vein endothelial cells derived from normal human newborns, cells derived from a single donor. EGM TM -2 BulletKit TM (CC-3162, LONZA):EBM TM -2 When the basal medium (CC-3156) is 500 mL, it contains VEGF (0.5 mL), hEGF (0.5 mL, human-derived EGF), R3-IGF-1 (0.5 mL), ascorbic acid (0.5 mL), hydrocortisone (0.2 mL), hFGFβ (2 mL, human-derived FGF beta), heparin (0.5 mL), FBS (10 mL), and GA (0.5 mL). Proteoglycan (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133)): Proteoglycan derived from salmon nasal cartilage. The GAG ​​used in Experiment 1 was prepared by purifying only GAG from this proteoglycan using standard methods. Unlike proteoglycans, GAG does not have a core protein attached to it. Fluorescently labeled proteoglycan No. 1 (also referred to as "labeled PG1" in the specification and drawings): ATTO 488 NHS ester (Sigma-Aldrich) and proteoglycan were reacted for 2 hours at room temperature according to the manufacturer's recommended method (method shown in the product: ATTO 488 NHS ester (Sigma-Aldrich)). After this reaction, the fluorescently labeled proteoglycan was purified using a Zeba Spin Desalting Column (Thermo Fisher Scientific) and recovered. Labeled PG1 is this recovered fluorescently labeled proteoglycan. Fluorescently labeled proteoglycan 2 (also referred to as "labeled PG2" in the specification and drawings): Atto 590 (Sigma-Aldrich, 70425) and proteoglycan were reacted for 2 hours at room temperature according to the manufacturer's recommended method (method shown in the product description for Atto 590 (Sigma-Aldrich, 70425)). After this reaction, the fluorescently labeled proteoglycan was purified using a Zeba Spin Desalting Column (Thermo Fisher Scientific) and recovered. Labeled PG2 is the recovered fluorescently labeled proteoglycan. Matrigel (Corning, Product Number: 356231): Corning TM Matrigel basement membrane matrix growth factor reduced phenol red free. Proteoglycan from bovine nasal septum (Sigma-Aldrich, product number: P5864): Proteoglycan from bovine nasal septum (chromatographically purified)

[0071] [Experiment 6: Evaluation of angiogenic potential using aortic ring assay] To confirm the effects of the unit containing VEGF, a collagen gel, and proteoglycans, an aortic ring assay (ex vivo assay) was performed using the thoracic aorta of 12-week-old male vascular reporter mice (Flt1-tdsRed BAC transgenic mice). The assay was performed according to the description in the paper (THE JOURNAL of JAPANESE COLLEGE of ANGIOLOGY, Vol. 45, No. 10, pp. 637-641).

[0072] First, a collagen gel layer (base layer) was prepared on a glass-bottom dish.

[0073] Next, a ring-shaped aortic slice (approximately 2 mm thick) was prepared for use in the experiment by removing the thoracic aorta from the reporter mouse and removing the adventitia from the aorta.

[0074] Next, approximately two aortic slices were placed on the dish perpendicular to the bottom of the culture dish, and a reconstituted collagen solution (0.3% by mass Cellmatrix type 1-A, Nitta Gelatin) was layered on top of it. After layering, the mixture on the dish (base layer, aortic slices, and reconstituted collagen solution) was allowed to stand at 37°C for 30 minutes to gel.

[0075] After the gelation, a control culture medium or a culture medium containing proteoglycan was added to the mixture. After the addition, the mixture to which the culture medium was added was cultured at 37°C for 14 days. The group to which the control culture medium was added was designated the "control group," and the group to which the proteoglycan-containing culture medium was added was designated the "PG-administered group." Cells extending from the aortic slices were observed using a phase-contrast microscope. The total area of ​​cells extending from the aortic slices (indicated by arrows in Figure 11) was measured for the control and PG-administered groups. The bar in Figure 11 indicates 200 μm. The total area value was used as a measurement of angiogenic activity.

[0076] The measurement results are as follows. When the total area of ​​the control group was set to 100, the area of ​​the PG-administered group was 555.2 (Student's t-test, p<0.001) compared to the control group. Therefore, the PG-administered group demonstrated significantly greater angiogenic potential than the control group.

[0077] In Experiment 6, the following samples were used: Collagen Gel: Collagen Gel Culturing Kit cellmatrix type 1-A (Nitta Gelatin, 638-00781) Glass bottom dish: 35mm glass bottom dish (IWAKI, 3911-035) Control culture medium (control group culture medium): In the case of 500 mL of basal medium, the 6 types of solutions contained in the HuMedia-EG growth additive set and VEGF (Peprotech, 3624436) at a final concentration of 20 ng / mL were added to the basal medium, but no proteoglycan was added. Culture medium containing proteoglycan (culture medium for PG administration group): In the case of 500mL of basal medium, this was a solution containing the six types of solutions contained in the HuMedia-EG growth additive set, VEGF (Peprotech, 3624436) to a final concentration of 20ng / mL, and proteoglycan at a final concentration of 100μg / mL. Basal medium: HuMedia-EB2 (Kurabo Industries, KE-2350S) HuMedia-EG Growth Additive Set (Kurashiki Boseki Co., Ltd., KE-6150): A growth additive kit for normal human vascular endothelial cells (for 500 ml) containing FBS (10 ml), hEGF (0.5 ml, human-derived EGF), hydrocortisone (0.5 ml), hFGFβ (0.5 ml, human-derived FGF beta), heparin (0.5 ml), and an antibiotic (0.5 ml, gentamicin / amphotericin B). Proteoglycan (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133)): Proteoglycan, derived from salmon nasal cartilage.

[0078] [Experiment 7: Evaluation of nerve and / or blood vessel regeneration potential using a sciatic nerve injury model] Using a mouse model with injured sciatic nerves (a mouse model with a severed sciatic nerve), we evaluated the regeneration ability of nerves and / or blood vessels by administering a unit containing VEGF, a gel component (Matrigel), and proteoglycan.

[0079] The experimental method is described below. First, the sciatic nerves (right and left sciatic nerves) near the tail of 12-week-old male vascular reporter mice (Flt1-tdsRed BAC transgenic mice) were cut to create model mice (n = 2). The right sciatic nerve of the model mice was treated with 10 mg / mL PG, and the left sciatic nerve of the model mice was treated with control.

[0080] Here, the following definitions are made. 10 mg / mL PG group treatment: A unit containing proteoglycan (final concentration 10 mg / mL) in Matrigel was placed on the sciatic nerve (the site of injury (cut) of the sciatic nerve). Control group treatment: Matrigel was placed on the sciatic nerve (the site of injury (cut) of the sciatic nerve). Proteoglycan (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133)): Proteoglycan, derived from salmon nasal cartilage. Matrigel (Corning, Product Number: 356231): Corning TM Matrigel basement membrane matrix growth factor reduced phenol red free.

[0081] Immediately after the treatment, the muscle layer and skin of the treated model mice were sutured. After the suture, the model mice were kept in normal conditions for 5 days or 8 days. After the normal conditions, the model mice were dissected and the treated area was examined with the naked eye and under a stereomicroscope. After the examination, a sciatic nerve (approximately 5 mm) was collected from the model mice.

[0082] The results of this confirmation and a photograph of the collected sciatic nerve are shown in FIG. In this confirmation, sciatic nerve regeneration was confirmed 5 days after treatment in the 10 mg / mL PG group (labeled 10 mg / mL PG in Figure 12) compared to the control group treatment (labeled Control in Figure 12, a group not containing proteoglycan) (the area indicated by the arrow in Figure 12). On the 8th day of treatment, sciatic nerve regeneration was further confirmed in the 10 mg / mL PG group treatment compared to the control group treatment (a group not containing proteoglycan). Furthermore, no inflammation was observed with the naked eye near the regenerated area. When the collected sciatic nerves were examined, it was confirmed that the sciatic nerves in the 10 mg / mL PG group were larger than those in the control group (group not containing proteoglycan) on both the 5th and 8th days of treatment.

[0083] [Experiment 8: Evaluation of angiogenesis using a sciatic nerve injury model] Using a mouse model with injured sciatic nerves (a mouse model with a severed sciatic nerve), we evaluated the vascular regeneration ability of administration of a unit containing a gel component (Matrigel) and proteoglycan using in vivo imaging. The evaluation was performed using the IVIS Imaging System (Perkin Elmer).

[0084] The experimental method is as follows. First, the sciatic nerves (right and left sciatic nerves) near the tail of 9-week-old female vascular reporter mice (Flt1-tdsRed BAC transgenic mice) were cut to create the model mice (n=1). The right sciatic nerve of the model mice was treated with 10 mg / mL PG, and the left sciatic nerve of the model mice was treated with the control group.

[0085] Here, the following definitions are made. PG group treatment: A unit containing proteoglycan (final concentration 10 mg / mL) in Matrigel was placed on the sciatic nerve (the site where the sciatic nerve was injured (cut)). Control group treatment: Matrigel was placed on the sciatic nerve (the site where the sciatic nerve was injured (cut)). Proteoglycan (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133)): Proteoglycan, derived from salmon nasal cartilage. Matrigel (Corning, Product Number: 356231): Corning TM Matrigel basement membrane matrix growth factor reduced phenol red free.

[0086] Immediately after the treatment, the muscle layer and skin of the treated model mice were sutured. After the suture, the mice were fed alfalfa-free fluorescent imaging diet (5V5M, PicoLab R The model mice were also kept in a normal condition for 7 days using SelectMouse50IF / 9F.

[0087] On the seventh day of the breeding, AngioSense TM AngioSense 750EX (Perkin Elmer, NeV10011EX) was administered via tail vein injection to the model mice (dose: 2 nmol / 100 μL of AngioSense 750 EX per mouse). Normal care was continued for a certain period of time after the injection. IVIS images of the model mice were taken 18, 21, and 24 hours after the injection.

[0088] The conditions for the shooting are as follows: Exposure: 5.00 seconds Bining: Medium Fstop: 2 Excitation: 640 Emission: Cy5.5

[0089] Just before the imaging, the model mouse was subjected to general anesthesia, and after the anesthesia, the body hair was removed from a predetermined area of ​​the model mouse. After the removal, the imaging was performed. The general anesthesia was performed by placing the model mouse for a certain period of time in a suction anesthesia box in which anesthesia was circulated. The removal involved the removal of body hair near the area where the above-mentioned treatment (PG group treatment or control group treatment) was performed.

[0090] The photographed results are shown in Figure 13. In Figure 13, (1) is the result after 18 hours, (2) is the result after 21 hours, and (3) is the result after 24 hours. The right side of Figure 13 shows the degree of fluorescence (Epi-fluorescence shown in Figure 13). This degree was calculated using the radiant efficiency (formula shown in Figure 13, radiant efficiency). In this degree, yellow indicates that more angiogenesis is occurring in the mouse body. Compared to the control treatment group (labeled Control in Figure 13), the PG treatment group (labeled PG in Figure 13) showed significant yellow expression (at the site of the arrow in Figure 13). This expression indicates the occurrence of angiogenesis.

[0091] In the evaluation shown in FIG. 13, the total radiant efficiency ([p / s] / [μW / cm 2 ], luminous intensity of the light source (light intensity) and Average Radiant Efficiency ([p / s / cm 2 / sr] / [μW / cm 2 ], the average luminance on the skin surface irradiated from the light source) was also quantified. The quantification results are shown in Tables 4 and 5. The labels "(1), (2), (3), Control, PG" in Tables 4 and 5 indicate the groups labeled in Figure 13. The quantification results also showed that the PG treatment group exhibited a significantly greater yellow color than the control treatment group.

[0092] [Table 4]

[0093] [Table 5]

[0094] [Experiment 9: Evaluation of angiogenesis, etc. using a sciatic nerve injury model] Using a mouse model with injured sciatic nerves (a mouse model with a severed sciatic nerve), we confirmed by immunohistochemical staining whether angiogenesis and nerve regeneration would occur when a unit containing gel components (Matrigel) and proteoglycan was administered.

[0095] The experimental method is as follows. First, the sciatic nerves (right and left sciatic nerves) near the tail of 12-week-old female vascular reporter mice (Flk1-GFP-BAC transgenic mice) were cut to create the model mice (n=1). The right sciatic nerve of the model mice was treated with 10 mg / mL PG, and the left sciatic nerve of the model mice was treated with the control group.

[0096] Here, the following definitions are made. PG group treatment: A unit containing proteoglycan (final concentration 10 mg / mL) in Matrigel was placed on the sciatic nerve (the site where the sciatic nerve was injured (cut)). Control group treatment: Matrigel was placed on the sciatic nerve (the site where the sciatic nerve was injured (cut)). Proteoglycan (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133)): Proteoglycan, derived from salmon nasal cartilage. Matrigel (Corning, Product Number: 356231): Corning TM Matrigel basement membrane matrix growth factor reduced phenol red free.

[0097] Immediately after the treatment, the muscle layer and skin of the treated model mice were sutured. After the suture, the mice were fed alfalfa-free fluorescent imaging diet (5V5M, PicoLab RThe model mice were housed under normal conditions for 8 days using a SelectMouse50IF / 9F strain. After the 8 days of normal housekeeping, the sciatic nerves from the right and left limbs of the model mice were harvested. The right limb was treated with 10 mg / mL PG, and the left limb was treated with the control.

[0098] The collected right and left sciatic nerves were washed with PBS for 5 minutes. After washing, the cells were fixed in 4% paraformaldehyde for 1 hour. After fixation, the medium was replaced with 30% sucrose solution. The right and left sciatic nerves were embedded in a frozen tissue sectioning embedding medium (Tissue-Tec OCT Compound, Sakura Finetech Japan) to prepare samples. The embedded samples were then stored at -80°C for 3 hours. Frozen sections of the right and left sciatic nerves were prepared using a cryostat HM525NX (Thermo Fisher Scientific, Inc.). The frozen sections were cut to 100 μm thickness.

[0099] The 100 μm-thick sections were blocked with blocking buffer at room temperature (approximately 25°C) for 3 hours. After blocking, the sections were subjected to primary antibody reaction overnight (approximately 8 hours) at 4°C in a reaction solution (primary antibody reaction solution) prepared by diluting a specific antibody with 10% donkey serum / PBS. After the primary antibody reaction, the sections were washed with 0.3% TritonX-100 / PBS (5 minutes x 3 times at room temperature). After washing, the washed sections were subjected to secondary antibody reaction at room temperature for 3 hours in a reaction solution (secondary antibody reaction solution) prepared by diluting a specific secondary antibody with 10% donkey serum / PBS. After the secondary antibody reaction, the sections were washed with 0.2% TritonX-100 / PBS (5 minutes x 1 time at room temperature). The washed sections were mounted using a specific mounting agent. The embedded sections were examined at 4x magnification using a confocal microscope (Olympus Corporation: Confocal Laser Scanning Microscope FV3000) to confirm whether angiogenesis and nerve regeneration had occurred.

[0100] The composition of the solution used to prepare the above-mentioned slices is as follows.

[0101] [Table 6]

[0102] [Table 7]

[0103] [Table 8]

[0104] [Table 9]

[0105] The primary antibody reaction was carried out using the primary antibody reaction solution shown in Table 10 below. [Table 10]

[0106] The secondary antibody reaction was carried out using the secondary antibody reaction solution shown in Table 11 below. [Table 11]

[0107] The results of Experiment 9 are shown in Figure 14. In Figure 14, "VEGFR2-EGFP" is the experimental group used to see whether angiogenesis was occurring (confirmed by the green expression of EGFP), "Neuro filament" is the experimental group used to see whether nerve regeneration was occurring (confirmed by the red expression), and "Merge" represents "VEGFR2-EGFP" and "Neuro filament" combined.

[0108] As shown in "VEGFR2-EGFP," significant green expression (indicated by the arrow in Figure 14) was observed in the PG treatment group (labeled PG in Figure 14) compared to the control treatment group (labeled Control in Figure 14). This expression indicates the occurrence of angiogenesis. As shown in "Neuro filament," the PG treatment group showed significantly higher red expression (arrow in Figure 14) than the control treatment group. This expression indicates that nerve regeneration (axon formation) is occurring. As shown in "Merge," it was confirmed that the areas where angiogenesis was occurring and the areas where nerve regeneration was occurring overlapped in the PG treatment group.

[0109] [Experiment 10: Evaluation of motor function using a sciatic nerve injury model] Using a mouse model with damaged sciatic nerves (a mouse model with a severed sciatic nerve), we evaluated whether motor function improved by administering a unit containing gel components (Matrigel) and proteoglycan using BBB scoring.

[0110] BBB scoring is a type of open field test. BBB scoring is an evaluation method in which subjects (hereinafter referred to as the model mice treated with the control group and the model mice treated with 10 mg / mL PG in this Experiment 10) are released into a space approximately 40-60 cm square and their behavior is observed and evaluated. The score table for the BBB scoring performed in Experiment 10 is shown in Table 12.

[0111] [Table 12]

[0112] The experimental method is as follows: First, two 6-week-old female vascular reporter mice (Flt1-tdsRed BAC transgenic mice) were prepared. The sciatic nerves in the tails of the two mice (the sciatic nerves in the right and left limbs) were cut to create the model mice. One model mouse (n=1) was treated with the control group at the amputation site, and the remaining model mouse (n=1) was treated with 10 mg / mL PG at the amputation site.

[0113] Here, the following definitions are made. PG group treatment: A unit containing proteoglycan (final concentration 10 mg / mL) in Matrigel was placed on the sciatic nerve (the site where the sciatic nerve was injured (cut)). Control group treatment: Matrigel was placed on the sciatic nerve (the site where the sciatic nerve was injured (cut)). Proteoglycan (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133)): Proteoglycan, derived from salmon nasal cartilage. Matrigel (Corning, Product Number: 356231): Corning TM Matrigel basement membrane matrix growth factor reduced phenol red free.

[0114] Immediately after the treatment, the muscle layer and skin of the treated model mice were sutured. After the suture, the model mice were kept in the usual care and subjected to BBB scoring (counting the score based on Table 12). The results are shown in Table 12. In Table 11, "2 hours" indicates the score 2 hours after the suture, "1 day" indicates the score 1 day after the suture, "8 days" indicates the score 8 days after the suture, and "17 days" indicates the score 8 days after the suture.

[0115] [Table 13]

[0116] As shown in Table 13, compared to the control group, the PG group showed an improvement in motor function on "17 days" (the treated hind limbs were always observed to kick the ground vigorously with the toes, and the direction of the toes was parallel to the trunk both when touching the ground and when leaving the bed).

[0117] [Experiment 11: Evaluation of angiogenesis using a sciatic nerve injury model] Using a mouse model with injured sciatic nerves (a mouse model with a severed sciatic nerve), we evaluated the vascular regeneration ability of administration of a unit containing a gel component (Matrigel) and proteoglycan using in vivo imaging. The evaluation was performed using the IVIS Imaging System (Perkin Elmer).

[0118] The experimental method is described below. First, the sciatic nerves (right and left sciatic nerves) near the tail of 9-week-old female vascular reporter mice (Flt1-tdsRed BAC transgenic mice) were transected to create the model mice (n=1). The left sciatic nerve of the model mice was treated with 10 mg / mL PG, and the right sciatic nerve of the model mice was treated with PG + SU4312.

[0119] Here, the following definitions are made. PG group treatment: A unit containing proteoglycan (final concentration 10 mg / mL) in Matrigel was placed on the sciatic nerve (the site where the sciatic nerve was injured (cut)). PG+SU4312 group treatment: A unit containing proteoglycan (final concentration 10 mg / mL) and SU4312 (final concentration 10 μM) in Matrigel was placed on the sciatic nerve (the site where the sciatic nerve was injured (cut)). Proteoglycan (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133)): Proteoglycan, derived from salmon nasal cartilage. Matrigel (Corning, Product Number: 356231): Corning TM Matrigel basement membrane matrix growth factor reduced phenol red free. SU4312 (S8567, Sigma-Aldrich): VEGF receptor inhibitor

[0120] Immediately after the treatment, the muscle layer and skin of the treated model mice were sutured. After the suture, the mice were fed alfalfa-free fluorescent imaging diet (5V5M, PicoLab R The model mice were also kept in a normal condition for 7 days using SelectMouse50IF / 9F.

[0121] On the seventh day of the breeding, AngioSense TM AngioSense 750EX (Perkin Elmer, NeV10011EX) was administered via tail vein injection to the model mice (dose: 2 nmol / 100 μL of AngioSense 750 EX per mouse). Normal care was continued for a certain period of time after the injection. IVIS images of the model mice were taken 24 hours after the injection.

[0122] The conditions for the shooting are as follows: Exposure: 5.00 seconds Bining: Medium Fstop: 2 Excitation: 640 Emission: Cy5.5

[0123] Just before the imaging, the model mouse was subjected to general anesthesia, and after the anesthesia, the body hair was removed from a predetermined area of ​​the model mouse. After the removal, the imaging was performed. The general anesthesia was performed by placing the model mouse for a certain period of time in a suction anesthesia box in which anesthesia was circulated. The removal involved the removal of body hair near the area where the above-mentioned treatment (PG group treatment or control group treatment) was performed.

[0124] The photographed results are shown in Figure 15. The right side of Figure 15 shows the degree of fluorescence (Epi-fluorescence shown in Figure 15). This degree was calculated using the radiant efficiency (formula shown in Figure 15, radiant efficiency). In terms of this degree, yellow indicates that more angiogenesis is occurring in the mouse body. Compared to the PG treatment group (labeled PG in Figure 15), the PG + SU4312 treatment group (labeled PG + SU4312 in Figure 15) showed lower expression of yellow.

[0125] In the group shown in FIG. 15, the total radiant efficiency ([p / s] / [μW / cm 2 ]) and Average Radiant Efficiency ([p / s / cm 2 / sr] / [μW / cm 2 ]) was also quantified. The quantification results are shown in Table 14. The labels "PG, PG+SU4312" in the table refer to the groups in Figure 15. The quantification results also showed that the expression level of yellow was lower in the PG+SU4312 treatment group compared to the PG treatment group. Based on the results of the PG+SU4312 treatment group, it is thought that if the PG group also had VEGF, further angiogenesis would occur.

[0126] [Table 14]

[0127] [Experiment 12: Confirmation of aggrecan expression in mouse brain tissue] To understand how aggrecan (proteoglycan) functions during the process of brain angiogenesis, we investigated its expression. Aggrecan is the major structural proteoglycan present in the extracellular matrix of cartilage tissue. Its molecular weight exceeds 2,500 kDa and its structure is composed of 100-150 glycosaminoglycan (GAG) chains bound to a core protein. Versican is a large proteoglycan distributed in a wide range of tissues, with a complex structure containing many N- and O-linked glycans. The versican core protein has hyaluronic acid-binding ability and is responsible for the formation and maintenance of the extracellular matrix.

[0128] The experimental method for Experiment 12 is described below. Brains were collected from mice at embryonic day 15.5, embryonic day 17.5, 1.5 months of age, and 14.5 months of age. The mice used in Experiment 10 were male vascular reporter mice (Flt1-tdsRed BAC transgenic mice).

[0129] The collected brain was washed with PBS for 5 minutes. After washing, the cells were fixed with 4% paraformaldehyde for 1 hour. After fixation, the brain was embedded in 5% agarose gel. The embedded brain was cut using a vibratome to prepare 150 μm thick sections. The sections were blocked overnight with a PBS solution containing 0.3% Triton and 10% donkey serum. After blocking, the sections were subjected to a primary antibody reaction overnight with a reaction solution containing a specified antibody (primary antibody reaction solution). After the primary antibody reaction, the sections were washed with PBS, and the washed sections were subjected to a secondary antibody reaction overnight with a secondary antibody reaction solution. After the secondary antibody reaction, the sections were washed with PBS, and the washed sections were mounted using a specified mounting agent. The embedded sections were examined for aggrecan expression using a confocal microscope (Olympus Corporation: Confocal Laser Scanning Microscope FV3000) at magnifications of 10x (Figure 16), 40x (Figure 17), and 20x (Figure 18).

[0130] The results of Experiment 12 are shown in Figures 16 to 18. Figure 17 is a 4x magnification of the area indicated by the arrow in the E17.5 group in Figure 16. As shown in Figure 16, we confirmed that aggrecan is expressed in the vasculature and versican in the nervous system in the developing cerebral cortex. As shown in Figure 17, aggrecan was expressed in capillaries near the cortical plate. As shown in Figure 18, when comparing M1.5 and M14.5, aggrecan expression was observed to decrease with age in M14.5.

[0131] In Figures 16 to 18, the following notations are used. E15.5: Group of mouse brains from embryonic day 15.5 E17.5: Group of mouse brains from embryonic day 17.5 M1.5: group of brains from 1.5-month-old mice M14.5: group of brains from 14.5-month-old mice Aggrecan: Experimental group immunostained for aggrecan with anti-aggrecan antibody VEGFR1-DSRed: VEGFR1 was immunostained with anti-VEGFR1 antibody and DSRed (fluorescent protein) was detected. VEGFR2-EGFP: Experimental group in which VEGFR2 was immunostained with anti-VEGFR2 antibody and the corresponding staining was demonstrated using EGFP (enhanced green fluorescent protein). Versican: Experimental group immunostained with anti-versican antibody DAPI: DAPI stained experimental group ·CP: cortical plate SP: Subplate ·IZ:inter mediate zone SVZ: subventricular zone VZ: ventricular zone

[0132] In Experiment 12, the primary antibody reaction solution shown in Table 15 below and the secondary antibody reaction solution shown in Table 16 below were used.

[0133] [Table 15]

[0134] [Table 16]

[0135] [Experiment 13: Measurement of the number of neurospheres (NS) formed and observation of NS] To confirm that the cells cultured in the presence of FGF-2, EGF, and proteoglycan were neural stem cells, the number of NSs formed and the morphology of the NSs were measured.

[0136] (Neural stem cell culture procedure) The neural stem cell culture procedure (experimental procedure) performed in Experiment 13 is described below. Figure 19 shows a schematic diagram of this experimental procedure. Female mice were generated on day 14 of pregnancy using ICR mice manufactured by Japan SLC Co., Ltd. Then, fetuses were removed from the mice and dissociated cells were prepared from the cerebral cortex of the fetuses. For this dissociation, 0.05 w / v% trypsin (a product of Fujifilm Wako Pure Chemical Corporation (0.25 w / v% trypsin-1 mmol / l EDTA·4Na solution (containing phenol red) (product codes: 201-16945, 209-16941)) diluted with PBS) was used. Of these dissociated cells, 240,000 cells were used to create the following treatment groups (control, medium 1, medium 2, medium 3). These cells were divided into samples at 60,000 cells per sample. Cells were seeded into a 6-well plate (Thermo Fisher Scientific, CAT#140675) containing a specified liquid medium. 2 ml of the specified liquid medium was added per well, and 10,000 cells were evenly seeded into each well. After seeding, the cells were cultured at 37°C and 5% CO2 for 7 days. After 7 days of culture, the cell clusters formed in the 6-well plate for each treatment group were observed using a specified phase-contrast microscope (20x magnification). Cell clusters with a radius of 50 μm or greater were defined as neurospheres, and the number of such neurospheres was counted. For comparison with the observations after 7 days of culture, observations of each treatment group on day 0 of culture were also performed using a specified phase-contrast microscope (20x magnification).

[0137] The composition of the liquid medium for each administration group (control, medium 1, medium 2, medium 3) is as follows: DMEM / Ham F-12 (Nacalai Tesque, Inc., 08460-95) was used as the basal medium for all administration groups, and the composition of each component in 50 ml is shown below. (control) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) · No proteoglycan

[0138] (Medium 1) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) Proteoglycan final concentration 10μg / ml

[0139] (Medium 2) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) Proteoglycan final concentration 100μg / ml

[0140] (Medium 3) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) Proteoglycan final concentration 1000μg / ml

[0141] The components contained in each administration group (control, medium 1, medium 2, medium 3) were as follows: N2: N-2 Supplement (100X) (gibco:17502-048) B27:B-27 TM Plus Supplement Vitamin A Free (50X) (gibco:A35828-01) Heparin: Heparin sodium (Nacalai Tesque (product code: 17513-96, 17513-41, 17513-54) Antibiotics: Penicillin-streptomycin (WAKO: 168-23191) ·bFGF:bFGF(funakoshi:100-18B) EGF: EGF (funakoshi: 315-09) Proteoglycan: Proteoglycan, derived from salmon nasal cartilage (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133))

[0142] (Measurement results of the number of neurospheres (NS) formed in each administration group) The measurement results are shown in Figure 20. Figure 20 shows the percentage of NS formation (%), with the number of NSs formed in the control group set at 100%. Medium 1 was 117.317753376014%, Medium 2 was 134.677950940218%, and Medium 3 was 149.305508553623%. It was confirmed that the number of NSs formed increases with an increase in proteoglycan content. It was confirmed that Medium 2 and Medium 3 showed a statistically significant increase in the number of NSs formed compared to the control.

[0143] (NS observations) The observation results are shown in Figure 21. For comparison with the observation results after 7 days of culture ("Culture 7 days" in Figure 21), the observation results after 0 days of culture ("Culture 0 days" in Figure 21) are also shown. Compared to the control, many NS were confirmed in media 1 to 3. Note that the arrows in Figure 21 indicate some of the NS.

[0144] Therefore, it was confirmed that the inclusion of FGF-2, EGF, and proteoglycan resulted in the proliferation of a greater number of neural stem cells.

[0145] [Experiment 14: Dual luciferase assay (confirmation of Hes-1 transcription activity)] By confirming the transcriptional activity of Hes-1, a protein (transcription factor) that controls the maintenance of undifferentiated state of neural stem cells and their differentiation into astrocytes, we confirmed whether the above-mentioned neural stem cells (cells cultured in a medium containing FGF-2, EGF, and proteoglycans) were maintaining their undifferentiated state.

[0146] The procedure for this dual luciferase assay is described below. First, neural stem cells were prepared. Figure 22 shows a schematic diagram of this experimental procedure. Female mice were generated on day 14 of pregnancy using ICR mice manufactured by Japan SLC Co., Ltd. Then, fetuses were removed from the mice and dissociated cells were prepared from the cerebral cortex of the fetuses. For this dissociation, 0.05 w / v% trypsin (Fujifilm Wako Pure Chemical Corporation product: 0.25 w / v% trypsin-1 mmol / L EDTA·4Na solution (containing phenol red) (product numbers: 201-16945, 209-16941) diluted with PBS) was used. These dissociated cells were divided into approximately 60,000 cells per sample to prepare samples for the following seven treatment groups (control, medium 1, medium 2, medium 3, medium 4, medium 5, and medium 6).

[0147] Hes-1 cDNA and a reporter gene (luciferase gene) were transfected into these samples using a transfection reagent (Thermo Fisher Scientific, Neon® Transfection System). The Hes-1 cDNA and reporter gene (luciferase gene) were "pHes1(2.5k)-luc (Plasmid #43806)" manufactured by Addgene. After this transfection, the cells were cultured for one day at 37°C in 5% CO2 using liquid media for each treatment group (control, medium 1, medium 2, medium 3, medium 4, medium 5, medium 6). After one day of culture, each sample was cultured with luciferase substrate (Thermo Fisher Scientific, Neon® Transfection System). TM Transfection System) was added, and a detection system (Promega, GloMax TM The luciferase expression in each sample was monitored using a Discover Microplate Reader.

[0148] The liquid media used in this dual luciferase assay for each administration group (control, medium 1, medium 2, medium 3, medium 4, medium 5, medium 6) have the following compositions. The control, medium 1, medium 2, and medium 3 have the same compositions as the liquid media used in Experiment 1 above. Note that DMEM / Ham's F-12 (Nacalai Tesque, Inc., 08460-95) was used as the basal medium for all administration groups, but the following shows the composition of each component in 50 ml. Note that for this culture, Corning's Costar TM 24 wells were used.

[0149] (control) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) · No proteoglycan

[0150] (Medium 1) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) Proteoglycan final concentration 10μg / ml

[0151] (Medium 2) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) Proteoglycan final concentration 100μg / ml

[0152] (Medium 3) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) Proteoglycan final concentration 1000μg / ml

[0153] (Medium 4) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) · Contains no bFGF ·EGF 50μl (final concentration 10ng / ml) Proteoglycan final concentration 1000μg / ml

[0154] (Medium 5) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) No EGF Proteoglycan final concentration 1000μg / ml

[0155] (Medium 6) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 150μl (final concentration 30ng / ml) ·EGF 50μl (final concentration 10ng / ml) · No proteoglycan

[0156] The components contained in each of the administration groups (control, medium 1, medium 2, medium 3, medium 4, medium 5, medium 6) were as follows: N2: N-2 Supplement (100X) (gibco:17502-048) B27:B-27 TM Plus Supplement Vitamin A Free (50X) (gibco:A35828-01) Heparin: Heparin sodium (Nacalai Tesque (product code: 17513-96, 17513-41, 17513-54) Antibiotics: Penicillin-streptomycin (WAKO: 168-23191) ·bFGF:bFGF(funakoshi:100-18B) EGF: EGF (funakoshi: 315-09) Proteoglycan: Proteoglycan, derived from salmon nasal cartilage (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133))

[0157] The results of observing luciferase expression levels (Hes-1 transcriptional activity) in each well are described below. The expression level in the control group was set to 100, with the expression levels in Medium 1 being 123.83, Medium 2 being 131.24, Medium 3 being 159.51, Medium 4 being 113.85, Medium 5 being 119.67, and Medium 6 being 131.55. The higher the luciferase expression level, the higher the Hes-1 transcriptional activity, suggesting that the cells maintained the undifferentiated state of neural stem cells. Medium 6 was used as a positive control for cells maintaining the undifferentiated state of neural stem cells. Compared to Medium 6 (which contained no proteoglycans but contained three times the amount of bFGF compared to the control), Medium 1, Medium 2, and Medium 3 (cells cultured in media containing FGF-2, EGF, and proteoglycans) were confirmed to maintain the undifferentiated state of neural stem cells.

[0158] [Experiment 15: Measurement of the number of NS formation] We examined whether cells cultured in a medium containing GAGs instead of proteoglycans could form NS. In this experiment, GAGs, unlike proteoglycans, do not have core proteins attached to them.

[0159] The procedure for Experiment 15 is described below. Figure 23 shows a schematic diagram of the experimental procedure. Female mice were generated on day 14 of pregnancy using ICR mice manufactured by Japan SLC Co., Ltd. Then, mouse fetuses were removed from the mice and dissociated cells were prepared from the cerebral cortex of the mouse fetuses. For this dissociation, 0.05 w / v% trypsin (a product of Fujifilm Wako Pure Chemical Corporation (0.25 w / v% trypsin-1 mmol / L EDTA·4Na solution (containing phenol red) (product codes: 201-16945, 209-16941)) diluted with PBS) was used. Of these dissociated cells, 180,000 cells were used to create the following treatment groups (control, medium 2, medium 7). These cells were divided into samples at 60,000 cells per sample. They were seeded into a 6-well plate (Thermo Fisher Scientific, CAT#140675) containing the designated liquid medium. Two milliliters of the specified liquid medium was added to a 6-well plate, and 10,000 cells were evenly seeded into each well. After seeding, the cells were cultured for 6 days at 37°C in a 5% CO environment. After 6 days of culture, the cell clusters formed in the 6-well plates of each treatment group were observed using a specified phase-contrast microscope (20x magnification). Cell clusters with a radius of 50 μm or greater were defined as neurospheres, and the number of such neurospheres was counted. For comparison with the observations after 6 days of culture, observations of each treatment group on day 0 of culture were also performed using a specified phase-contrast microscope (20x magnification).

[0160] The liquid medium compositions for each administration group (control, medium 2, medium 7) are as follows. Control and medium 2 have the same composition as the liquid medium used in Experiment 1 above. Note that DMEM / Ham F-12 (Nacalai Tesque, Inc., 08460-95) was used as the basal medium for all administration groups, and the composition of each component in 50 ml is shown below. (control) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) · No proteoglycan

[0161] (Medium 2) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) Proteoglycan final concentration 100μg / ml

[0162] (Medium 7) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) ·GAG final concentration 100μg / ml

[0163] The components contained in each administration group (control, medium 2, medium 7) were as follows: N2: N-2 Supplement (100X) (gibco:17502-048) B27:B-27 TMPlus Supplement Vitamin A Free (50X) (gibco:A35828-01) Heparin: Heparin sodium (Nacalai Tesque (product code: 17513-96, 17513-41, 17513-54) Antibiotics: Penicillin-streptomycin (WAKO: 168-23191) ·bFGF:bFGF(funakoshi:100-18B) EGF: EGF (funakoshi: 315-09) Proteoglycan: Proteoglycan derived from salmon nasal cartilage (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133)). The GAG ​​used in Experiment 3 was prepared by purifying only GAG from this proteoglycan using standard methods.

[0164] The results of measuring the number of NSs formed are shown below. The percentage of NSs formed (%) is calculated by taking the number of NSs formed in the control group as 100%. The percentage for Medium 2 was 118.7%, and for Medium 7 it was 58.8%. This suggests that the structure of proteoglycan (the bond between the core protein and GAG structure) allows for the formation of NSs.

[0165] [Experiment 16: Confirmation of proteoglycan localization in neural stem cells] We investigated the localization of proteoglycans in neural stem cells grown in a medium containing proteoglycans.

[0166] The procedure for this confirmation is explained below, and a schematic diagram of this experimental procedure is shown in Figure 24. First, neural stem cells were prepared. Female ICR mice (Japan SLC Co., Ltd.) were bred on day 14 of pregnancy. Then, fetuses were removed from the mice and dissociated cells were prepared from the cerebral cortex of the fetuses. For this dissociation, 0.05 w / v% trypsin (Fujifilm Wako Pure Chemical Corporation, 0.25 w / v% trypsin-1 mmol / l EDTA·4Na solution (containing phenol red) (product numbers: 201-16945, 209-16941) diluted with PBS) was used. From these dissociated cells, 180,000 cells were used to prepare the following medium groups (Medium 2, Medium 8). These cells were divided into samples at 60,000 cells per sample. They were seeded into 6-well plates (Thermo Fisher Scientific, Inc., CAT#140675) containing the designated liquid medium. Two milliliters of the specified liquid medium (medium described in the corresponding medium group) was evenly seeded into each well of a 6-well plate so that 10,000 cells were present. After seeding, the cells were cultured at 37°C and 5% CO2 for 24 hours to proliferate the neural stem cells and prepare them for observation. One hour before collecting the neural stem cells to be used for observation, Hoechst 33342 (final concentration 10 mg / mL, Hoechst 33342) was added to the medium. TM The nuclei of neural stem cells were stained using the 33342 Imaging Protocol (Thermo Fisher Scientific). Observations were performed using a fluorescence microscope (Olympus Corporation: Inverted Research Microscope IX81) at 20x magnification to observe the localization of proteoglycans in the formed cells in the 6-well plates of each treatment group after 24 hours of culture.

[0167] The liquid medium compositions for each medium group (medium 2, medium 8) used in Experiment 16 are as follows. Medium 2 has the same composition as the liquid medium used in Experiment 1 above. Note that DMEM / Ham F-12 (Nacalai Tesque, Inc., 08460-95) was used as the basal medium for all treatment groups, and the composition of each component in 50 ml is shown below.

[0168] (Medium 2) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) Proteoglycan final concentration 100μg / ml

[0169] (Medium 8) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) Fluorescently labeled proteoglycan, final concentration 100 μg / ml

[0170] The components contained in each medium group (medium 2, medium 8) are as follows: The following was used. N2: N-2 Supplement (100X) (gibco:17502-048) B27:B-27 TM Plus Supplement Vitamin A Free (50X) (gibco:A35828-01) Heparin: Heparin sodium (Nacalai Tesque (product code: 17513-96, 17513-41, 17513-54) Antibiotics: Penicillin-streptomycin (WAKO: 168-23191) ·bFGF:bFGF(funakoshi:100-18B) EGF: EGF (funakoshi: 315-09) Proteoglycan: Proteoglycan, derived from salmon nasal cartilage (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133))

[0171] The "fluorescently labeled proteoglycan" contained in Medium 8 was prepared as follows: ATTO 488 NHS ester (Sigma-Aldrich) and proteoglycan were reacted for 2 hours at room temperature according to the manufacturer's recommended method (Product: ATTO 488 NHS ester (Sigma-Aldrich)). After this reaction, the fluorescently labeled proteoglycan was purified using a Zeba Spin Desalting Column (Thermo Fisher Scientific) and recovered. This recovered fluorescently labeled proteoglycan was used in Experiment 4.

[0172] The observation results are shown in Figure 25. Figure 25 shows the results of observation under a fluorescent microscope of the medium 2 group (a medium containing non-fluorescently labeled proteoglycan, unlike the medium 8 group) and the medium 8 group (a medium containing fluorescently labeled proteoglycan, unlike the medium 8 group). In Figure 7, "Hoechst" stands for Hoechst staining (Hoechst TM This shows that neural stem cell nuclei were labeled using the 33342 imaging protocol (Thermo Fisher Scientific), and "Fluorescein-PG" indicates that localized proteoglycans were labeled by fluorescent staining. In the medium 8 group, fluorescently labeled proteoglycans were confirmed to be localized around the neural stem cells. These results suggest that proteoglycans added to the neural stem cell culture medium are localized around the neural stem cells and may be involved in the construction of the pericellular membrane (PCM).

[0173] [Experiment 17: Confirmation of the localization of proteoglycans in neural stem cells (confocal microscopy)] In neural stem cells grown in a medium containing proteoglycans, the location of proteoglycans was also confirmed using a confocal microscope.

[0174] The procedure for this confirmation will be explained below. First, neural stem cells were prepared. Female ICR mice (Japan SLC Co., Ltd.) were bred on day 14 of pregnancy. Then, fetuses were removed from the mice and dissociated cells were prepared from the cerebral cortex of the fetuses. For this dissociation, 0.05 w / v% trypsin (Fujifilm Wako Pure Chemical Corporation, 0.25 w / v% trypsin-1 mmol / L EDTA·4Na solution (containing phenol red) (product numbers: 201-16945, 209-16941) diluted with PBS) was used. From these dissociated cells, 180,000 cells were used to prepare the following medium group (Medium 9). These cells were divided into 60,000 cells per sample and seeded into 6-well plates (Thermo Fisher Scientific, Inc., CAT#140675) containing the designated liquid medium. Two milliliters of the specified liquid medium (medium described in the corresponding medium group) was evenly seeded into each well of a 6-well plate so that 10,000 cells were present. After seeding, the cells were cultured at 37°C and 5% CO2 for 24 hours to proliferate the neural stem cells, and the neural stem cells used for observation were prepared. One hour before collecting the neural stem cells to be used for observation, Hoechst 33342 (final concentration 10 mg / mL, Hoechst 33342) was added to stain the nuclei of the neural stem cells. TM 33342 Imaging Protocol (Thermo Fisher Scientific)) and Cell mask (final concentration 5 mg / mL, CellMas) to stain the cell membrane of neural stem cells. TMThe cells were stained with a dye (Thermo Fisher Scientific), collected by centrifugation at 400 g for 3 minutes, and then fixed with 4% paraformaldehyde for 10 minutes. The cells were washed with PBS and mounted on glass slides with mounting medium to prepare specimens. The localization of neural stem cell nuclei, neural stem cell cell membranes, and proteoglycans was compared using a confocal microscope (Olympus Corporation: FV3000 Confocal Laser Scanning Microscope) at 40x magnification.

[0175] The liquid medium compositions of each medium group (medium 9) used in Experiment 17 are as follows: DMEM / Ham F-12 (Nacalai Tesque, Inc., 08460-95) was used as the basal medium, and the composition of each component in 50 ml is shown below.

[0176] (Medium 9) N2 500μl B27 1ml ·Heparin 50μl (final concentration 2ng / ml) ·Antibiotic 100μl (Final concentrations: penicillin 100U / ml, streptomycin 10μg / ml) ·bFGF 50μl (final concentration 10ng / ml) ·EGF 50μl (final concentration 10ng / ml) Fluorescently labeled proteoglycan, final concentration 100 μg / ml

[0177] The components contained in this medium 9 are as follows: N2: N-2 Supplement (100X) (gibco:17502-048) B27:B-27 TM Plus Supplement Vitamin A Free (50X) (gibco:A35828-01) Heparin: Heparin sodium (Nacalai Tesque (product code: 17513-96, 17513-41, 17513-54) Antibiotics: Penicillin-streptomycin (WAKO: 168-23191) ·bFGF:bFGF(funakoshi:100-18B) EGF: EGF (funakoshi: 315-09) Proteoglycan: Proteoglycan, derived from salmon nasal cartilage (Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 162-22131, 168-22133))

[0178] The "fluorescently labeled proteoglycan" containing Medium 9 was prepared as follows. Atto 590 (Sigma-Aldrich, 70425) and proteoglycan were reacted for 2 hours at room temperature according to the manufacturer's recommended method (Product: Atto 590 (Sigma-Aldrich, 70425)). After this reaction, the fluorescently labeled proteoglycan was purified using a Zeba Spin Desalting Column (Thermo Fisher Scientific) and recovered. This recovered fluorescently labeled proteoglycan was used in Experiment 5.

[0179] The observation results are shown in Figure 26. "Hoechst" indicates that the nuclei of neural stem cells were labeled with Hoechst staining, and "Cell mask" indicates that the nuclei were labeled with CellMask. TM The staining indicates that the neural stem cell membrane was labeled, "PG-Atto590" indicates that proteoglycans were labeled, and "Merge" indicates the combined labeling of the nucleus, the cell membrane, and the proteoglycan. The results shown in Figure 26, similar to those shown in Figure 25, suggest that the proteoglycans added to the neural stem cell culture medium are localized around the neural stem cells and may be involved in the construction of the pericellular membrane (PCM).

[0180] The above has described the embodiments (including examples) of the present invention with reference to the drawings, but the specific configuration of the present invention is not limited to this, and even if there are design changes, etc., within the scope that does not deviate from the gist of the present invention, they are included in the present invention.

Claims

1. A gel component and a proteoglycan are included, The proteoglycan is derived from salmon nasal cartilage or bovine nasal septum, A unit used for promoting angiogenesis, wherein the content of the proteoglycan is 0.1 μg / ml or more and 1000 μg / ml or less.

2. The unit of claim 1 comprising VEGF.

3. The unit according to claim 1 or 2, which contains FGF-2.

4. The unit according to any one of claims 1 to 3, which contains EGF.

5. A unit described in any one of claims 1 to 4, further comprising cells selected from the group consisting of cardiomyocytes, endothelial cells, mural cells, and muscle cells.

6. The unit according to any one of claims 2 to 5, wherein a liquid medium containing VEGF and cells selected from the group consisting of cardiomyocytes, endothelial cells, mural cells, and muscle cells are layered on top of the gel component.

7. The unit according to any one of claims 2 to 6, wherein the proteoglycan is contained in a liquid medium containing VEGF.

8. The unit according to any one of claims 1 to 7, wherein the proteoglycan is contained in a gel component.

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