Patent Application
20230138007
Embodiments relate to a unit for promoting angiogenesis and/or nerve regeneration in humans and the like.
Angiogenesis is a phenomenon in animals in which vascular endothelial cells migrate and proliferate from existing blood capillaries and other vessels to form a new vascular network through lumen formation. During the wound healing process (inflammatory, proliferative, and maturation phases), new blood vessels are formed toward the injured area to transport oxygen and energy necessary for the healing of the injured area. Therefore, it is thought that wound healing can be accelerated by promoting angiogenesis.
Vascular endothelial growth factor (VEGF), for example, is known as to induce angiogenesis. However, VEGF is known to be produced by tumor cells, for example, and induces pathological angiogenesis (Patent Literature 1). There is also technology to generate endothelial cells from human induced pluripotent stem cells (iPS cells) (see Patent Literature 2). However, there is a possibility that transplantation of iPS cells with remaining undifferentiated cells may cause cancer, and there are safety issues with the use of this technology.
In addition, the transplantation of neural stem cells for the treatment of neurological diseases such as Parkinson's disease and spinal cord injury is also being considered due to a lack of donors and ethical issues (Non-Patent Literature 1, Patent Literature 3).
With the discovery of iPS cells, it is now possible, for example, to create iPS cells from the patients themselves and to induce neural stem cells from them. It is now expected that autologous transplantation, in which the patient's own cells can be used for treatment. However, it is believed that it takes more than half a year to create neural stem cells from the patient's own cells via iPS cells. In addition, the safety and direction of differentiation of iPS cell lines are considered to differ greatly from one cell line to another. It takes even longer to verify that an iPS cell is safe. Furthermore, in spinal cord injury, neural stem cells are considered to be ineffective unless they are transplanted before the patient's symptoms become fixed. Therefore, for example, in such diseases where cell transplantation is effective for a limited period, it would be difficult to use the patient's own cells for treatment even if iPS cell transplantation technology is used (Non-Patent Literature 1). In addition, even in the case of transplantation of such neural stem cells, the cell viability after such transplantation may be low (Non-Patent Literature 2).
There is still a need for safe means of promoting angiogenesis and/or nerve regeneration (e.g., techniques to induce angiogenesis in cells and tissues transplanted into the body), development of scaffolds and other agents to allow neural stem cells (undifferentiated tissue stem cells of the nervous system) to grow and proliferate after such transplantation, and the like.
Therefore, the object of embodiments is to provide a safe means of promoting angiogenesis, and the like.
The embodiments were made to solve such problems, i.e., one aspect of the embodiments is “a unit for use in promoting angiogenesis and/or nerve regeneration, comprising a gel component and proteoglycans,” and the embodiments include, for example, the following (1) to (11).
By using this unit, for example, it is possible to safely promote angiogenesis in a human body by transplanting this unit into the body.
The following is a description of the units involved in the embodiments
Vascular endothelial growth factor (VEGF) is a growth factor that specifically affects vascular endothelial cells isolated from culture media of pituitary folliculo-stellate cell. VEGF is a member of the cysteine-knot growth factor superfamily and is a cell growth factor that plays an important role in de novo angiogenesis (vasculogenesis) during the embryonic period, and the formation of new blood vessels by branching and elongation of existing vessels (angiogenesis). VEGF is a member of the VEGF family of proteins, which includes VEGF-A, VEGF-B, and the like. VEGF-A has effects on vascular endothelial cell proliferation, enhancement of vascular permeability, promotion of ductal vaginogenesis, and induction of production of active substances from endothelial cells, while VEGF-E has specific effects on tumor tissue angiogenesis.
Liquid medium is preferred for the medium used in the embodiments in terms of simplicity of medium preparation (e.g., it is considered easier to prepare the medium when performing floating culture). The liquid medium used can be any known medium. For example, a medium containing the components (inorganic salts, carbohydrates, hormones, essential amino acids, and vitamins) necessary for cell survival and growth (e.g. Iscove modified Dulbecco's medium (IMDM), RPMI, and DMEM (e.g., DMEM/Ham's F-12 medium (NACALAI TESQUE, INC. 08460-95) used in the following examples), Fischer's medium, a-medium, Leibovitz medium, L-15 medium, NCT L-15 medium, NCTC medium, F-12 medium, MEM, and McCoy medium). Further additives (serum, antibiotics, etc.) may also be included as needed.
Angiogenesis is an important morphogenetic phenomenon that creates a network of new blood vessels in vivo, in which vascular endothelial cells collaborate with other cells to promote the formation of new blood vessels in a wound-specific manner. (In more detail, vascular endothelial cells cooperate with other cells to form characteristic vascular structures, such as two- and three-dimensional branching structures, through repeated budding, elongation, branching, and lumen formation.) In vivo phenomena that occur in normal (physiological) angiogenesis include fetal angiogenesis, endometriogenesis, follicle formation, and wound healing. In vivo phenomena that occur with pathological angiogenesis are, for example, malignant tumor formation, intraocular angiogenic disease, rheumatoid arthritis, and atherosclerosis. In vivo phenomena caused by inadequate angiogenesis include, for example, arteriosclerosis obliterans, angina pectoris, and myocardial infarction. To treat in vivo phenomena (diseases) caused by inadequate angiogenesis, vascular regeneration therapy is used, for example, to promote angiogenesis.
Cells to cause angiogenesis promotion used in the embodiments are, any kind of mammalian cells, such as human and other mammalian cells, for example, somatic cells, progenitor cells of such somatic cells, or mixed cells thereof. Cells for causing angiogenesis promotion used in the embodiments include, for example, cardiomyocytes, endothelial cells (vascular endothelial cells, lymphatic endothelial cells, etc.), wall cells (pericytes, etc.), muscle cells (skeletal muscle cells, smooth muscle cells, etc.), etc., as somatic cells. In the following examples, HUVECs (human umbilical vein endothelial cells) were used as cells to promote angiogenesis.
The gel component (material) used in the embodiments is usually biocompatible and can contain large amounts of water, allowing easy diffusion and transfer of substances necessary for cell survival, such as oxygen, water, nutrients and enzymes. The form of the gel component is not limited as long as it can be incorporated into the unit, for example, particulate, granular, film, tube, disk, mesh, mesh, porous, suspended or dispersed. Gel components include, for example, hydrogels such as gelatin hydrogels. The gel component can also contain various components, such as laminin, type IV collagen, entactin/nidogen, and predetermined growth factors, as needed to adjust the proliferative potential of cells to promote angiogenesis. In the examples below, the gel component is, for example, Matrigel (registered trademark, Corning, Product Number: 356231), and Collagen Gel Culturing Kit cellmatrix typel-A (Nitta Gelatin, 638-00781) was used.
Proteoglycans are glycoproteins with sugar chains called glycosaminoglycans such as chondroitin sulfate and dermatan sulfate (hereinafter referred to as GAGs) covalently attached to the core protein. Proteoglycans are widely distributed in the body, including skin and cartilage, as one of the major components of the extracellular matrix. GAG chains have a long linear structure with no branches. Because of the large number of sulfate and carboxy groups, it is negatively charged, and the GAG chain takes on an extended shape due to its electrical repulsive force. In addition, proteoglycans can hold a large amount of water due to the water affinity of sugars. The large number of GAG chains in proteoglycans is responsible for the unique functions of cartilage, such as elasticity and resistance to impact, while flexibly holding water like a sponge.
The core protein of proteoglycans has the property of binding to various molecules in the matrix. In the case of cartilage proteoglycans, the N-terminus has a binding region for hyaluronan and link protein, which can bind to these substances, and also aggregate between the same molecules, and the C-terminus has lectin-like and EGF-like regions that can bind to various other molecules. These properties allow proteoglycans to develop structures that are unique to each tissue. Among proteoglycans, chondroitin sulfate-type proteoglycans are proteoglycans in which chondroitin sulfate is covalently bound to the core protein.
Proteoglycans derived from salmon nasal cartilage are proteoglycans extracted from salmon nasal cartilage. Here, salmon is, for example, a member of the genus Oncorhynchus, preferably with the scientific name “Oncorhynchus” in terms of cell proliferation (e.g., more efficient cultivation of the cells to be cultured). “Oncorhynchus keta” is selected for its cell proliferation (e.g., more efficient cultivation of the cells to be cultured).
The content of proteoglycans in the unit of the embodiments, for example, in terms of cell proliferative properties, has a lower limit of preferably 0.1 μg/ml or more, more preferably 0.2 μg/ml or more, and still more preferably 0.5 μg/ml or more.
The proteoglycans contained in the unit of the embodiments are prepared by the method described in, for example, the gazette (Japanese Patent No.6317053). In the experiments shown in the following examples, proteoglycans derived from salmon nasal cartilage (FUJIFILM Wako Pure Chemical Corporation (product codes: 162-22131, 168-22133)), proteoglycans derived from bovine nasal septum and the like were used.
In the units of the embodiments, proteoglycans are included in the liquid medium and/or gel component.
As described above, the unit of the embodiments is “unit (population) used for angiogenesis promotion, including a gel component and a proteoglycan”, “unit (population) containing a liquid medium containing VEGF, cells to cause angiogenesis promotion, a gel component, and a proteoglycan” and the like. For example, it is also possible to create multiple stacked units by using further gel component, and transplant the stacked unit into human body or the like.
The unit of the embodiments can be used, for example, in cell therapy such as cell transplantation (a therapy to treat diseases using one's own cells or cells of others), regenerative medicine (a therapy to induce functional tissue regeneration by local mobilization of stem cells existing in vivo to injured or diseased tissue without removing them from the body).
FGF-2 (Fibroblast Growth Factor-2, also called bFGF) can be isolated from animal nervous tissue, pituitary gland, adrenal cortex, and placenta. 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, such as a cell growth factor, angiogenic factor, and neurotrophic factor, and shows proliferative activity against a variety of cells, including ES cells and iPS cells.
EGF (Epidermal Growth Factor) is a polypeptide that promotes the proliferation of various cells including epithelial cells. EGF is not species specific and has proliferative effects on a variety of cells including epithelial cells, fibroblasts, and hepatocytes. In vivo, EGF is considered to be a growth factor that plays an important role in cell proliferation and differentiation.
Neural stem cells are undifferentiated tissue stem cells of the nervous system that combine self-renewal and pluripotency. In the embryonic brain of humans and other mammals, neural stem cells first proliferate actively, increasing their number exponentially, and then generating asymmetric division. It is also known to produce astrocytes and oligodendrocytes in the late fetal and postnatal brain. Neural stem cells are the source of neurons, astrocytes, and oligodendrocytes, the major cell types that make up the central nervous system. In the embodiments, preferably, cells from mammals such as humans, mice, and the like are used.
Nerve regeneration, such as of central and/or peripheral nerves, represents at least partial reproduction of the process of normal development in nerves and is independent of the origin of the cells to be regenerated. Cells to be regenerated include, for example, stem cells (e.g., neural stem cells, embryonic stem cells, bone marrow cells, etc.), neural progenitor cells, or neurons. Furthermore, the nerve-regenerating cells may be intrinsic (e.g., neural stem cells, neural progenitor cells, neurons, mature neurons, etc.) or exogenous (e.g., transplanted neural stem cells, transplanted neural progenitor cells, transplanted neurons, transplanted mature neurons, etc.). Exogenous cells may be autologous cells or cells derived from other families. Neural regeneration encompasses tissue regeneration or functional regeneration, and includes, for example, cell viability, differentiation, proliferation and/or maturation as described above. Maturation is, for example, the growth of nerve cells to a functionally working state such as signal exchange. Regeneration also encompasses, for example, neurotrophic factor-like effects and enhanced neurotrophic factor activity. For example, peripheral nerve regeneration is the extension of peripheral nerves injured by external or internal factors to target cells, the ability to reconstruct nerve circuits, and the prevention and/or treatment of peripheral nerve disorders.
Umbilical vein endothelial cells derived from human normal newborns (Human Umbilical Vein Endothelial Cells (HUVECs)) were used to evaluate the angiogenic potential of units containing proteoglycans and other substances.
The experimental procedure is described below. First, Matrigel (150 μl/well) was placed in each well and the wells were incubated at room temperature (23° C.) for 10 minutes. After this incubation, the wells were further incubated at 37° C. and 5% CO2 for 30 minutes. After this 30-minute incubation, culture medium (EGM™-2 BulletKit™) was added to each well and equilibration was performed at 37° C., 5% CO2 for 15 minutes. After the equilibration, HUVECs (5.0×104 cells/well) were inoculated to each well for the following groups (PG0, PG1, PG10, PG100, PG1000, and GAG1000) under the conditions of the culture medium at 37° C., 5% CO2. Two hours after the start of this culture (indicated as 2 h in
In this Experiment 1, the composition of the medium of each group (PG0, PG1, PG10, PG100, PG1000, and GAG1000) is as follows.
No proteoglycans in culture medium (EGM™-2 BulletKit™)
Culture medium (EGM™-2 BulletKit™) to which the proteoglycan was added at a final concentration of 1 μg/ml.
Culture medium (EGM™-2 BulletKit™) to which the proteoglycan was added at a final concentration of 10 μg/ml.
Culture medium (EGM™-2 BulletKit™) to which the proteoglycan was added at a final concentration of 100 μg/ml.
Culture medium (EGM™-2 BulletKit™) to which the proteoglycan was added at a final concentration of 1000 μg/ml.
Culture medium (EGM™-2 BulletKit™) to which GAG was added at a final concentration of 1000 μg/ml.
The results of this Experiment 1 are shown in
HUVEC was used to evaluate the angiogenic potential of the unit containing the proteoglycan and other substances. Unlike Experiment 1, in this Experiment 2, the angiogenic potential was evaluated by adding a predetermined amount of the proteoglycan to Matrigel.
The experimental procedure is described below. In each well, Matrigel (150 μl/well, without proteoglycan) or Matrigel with the proteoglycan added at 100 μg/ml (150 μl/well) were allowed to stand at room temperature (23° C.) for 10 minutes. Here, in
The results of this Experiment 2 are shown in
HUVECs were used to confirm the localization of proteoglycans in angiogenesis using fluorescence and confocal microscopy.
The procedure for this Experiment 3 is described below. First, prepare the following three types of wells.
After these wells were prepared, they were allowed to stand at room temperature (23° C.) for 10 minutes. After this incubation, the wells were further incubated at 37° C. and 5% CO2 for 30 minutes.
After this 30-minute incubation, the culture medium (EGM™-2 BulletKit™) was added to each well and equilibrated for 15 minutes at 37° C. and 5% CO2. After the equilibration, each well was inoculated with HUVECs (5.0×104 cells/well). Here, in the group to which Matrigel was added (70 μl/well) (The group of Labeled PG in medium), “Labeled PG1” was further added to the culture medium to reach a final concentration of 100 μg/ml in the unit of Matrigel and culture medium.
After the inoculation, cells were cultured for 2 hours at 37° C., 5% CO2. After the culture, cells in each well were collected by centrifugation at 400×g for 3 minutes. The collected cells were washed with PBS for 5 minutes. After the wash, the cells were fixed in 4% paraformaldehyde for 15 minutes. After the fixation, cells were again washed with PBS for 5 minutes. After the wash, the cells were mounted on glass slides with DAPI-containing encapsulating agent (Dojin Kagaku Kenkyusho Co., Ltd. D212-Cellstain™-DAPI) to prepare observation samples. Fluorescence microscopy (Olympus Corporation: Inverted Research Microscope IX81) and confocal microscopy (Olympus Corporation: Confocal Laser Scanning Microscope FV3000) were used at 40× magnification, to compare HUVEC nuclei (stained with DAPI), and the localization of proteoglycans.
Observations are shown in
Using HUVECs, the localization of proteoglycans in angiogenesis was confirmed using fluorescence and confocal microscopy. The labeled proteoglycan used in this Experiment 4 was not the labeled PG2 used in Experiment 3, but the labeled PG1.
The procedure for this Experiment 4 is described below. First, prepare the following two types of wells.
After these wells were prepared, they were allowed to stand at room temperature (23° C.) for 10 minutes. After this incubation, the wells were further incubated at 37° C., 5% CO2 for 30 minutes.
The culture medium (EGM™-2 BulletKit™) was added to each well and equilibrated for 15 minutes at 37° C. and 5% CO2. After the equilibration, each well was inoculated with HUVECs (5.0×104 cells/well). Here, in the group to which Matrigel was added (70 μl/well) (The group of Labeled PG in medium), “Labeled PG1” was further added to the culture medium to reach a final concentration of 100 μg/ml in the unit of Matrigel and culture medium.
After the inoculation, cells were cultured for 2 hours at 37° C., 5% CO2. One hour before collecting the cells to be used for observation (after the culture), the nuclei of the cells were stained with Hoechst 33342 (final concentration 10 mg/mL. Hoechst™ 33342 Imaging protocol (Thermo Fisher Scientific) and Cellmask (final concentration 5 mg/mL, the CellMask™ staining (Thermo Fisher Scientific) was added and stained. After the incubation, cells were collected by centrifugation at 400 g for 3 minutes. The collected cells were washed with PBS for 5 minutes. After the wash, the cells were fixed in 4% paraformaldehyde for 15 minutes. After the fixation, the cells were washed again with PBS for 5 minutes. After the wash, the cells were mounted on glass slides with an encapsulant to prepare observation specimens. A confocal microscope (Olympus Corporation: Confocal Laser Scanning Microscope FV3000) was used to compare the HUVEC nuclei (stained with Hoechst as described above), the HUVEC cell membrane (stained with Cellmask), and proteoglycan localization, with magnifications of 20×, 40× and 60×.
The observations are shown in
HUVECs were used to evaluate the angiogenic potential of the unit containing proteoglycans and other substances.
The experimental procedure is described below. First, Matrigel (150 μl/well) was placed in each well and the wells were allowed to stand at room temperature (23° C.) for 10 minutes. After this incubation, the wells were further incubated at 37° C., 5% CO2 for 30 minutes. After this 30-minute incubation, culture medium (EGM™-2 BulletKit™) was added to each well and equilibrated at 37° C., 5% CO2 for 15 minutes. After the equilibration, HUVECs (5.0×104 cells/well) were inoculated in each well for each of the following groups (PG0, PG100, bovine PG100), the cells were cultured under the conditions of 37° C., 5% CO2 medium. At 6 hours after the start of this culture, the specified evaluations were performed.
In this Experiment 5, the medium composition of each group (PG0, PG100 and bovine PG100) is as follows.
Culture medium (EGM™-2 BulletKit™) , without proteoglycan.
Culture medium (EGM™-2 BulletKit™) to which proteoglycan (proteoglycan derived from salmon nasal cartilage) are added at a final concentration of 100 μg/ml.
Culture medium (EGM™-2 BulletKit™) to which proteoglycans derived from bovine nasal septum are added at a final concentration of 100 μg/ml.
The results of this Experiment 5 are shown in
The results of Experiment 5 showed that not only the addition of proteoglycans derived from salmon nasal cartilage to the medium (PG100), but also the addition of bovine nasal septum-derived (bovine PG100), promoted angiogenesis.
The following samples and other materials were used in the Experiment 1 to Experiment 5.
To confirm the effects of the unit containing VEGF, gel components (collagen gel), and proteoglycans, thoracic aorta of 12-week-old male vascular reporter mice (Fltl-tdsRed BAC transgenic mice) were used to perform an aortic ring assay (ex vivo assay). The assay was published in an article (THE JOURNAL of JAPANESE COLLEGE of ANGIOLOGY Vol. 45 No. 10, pp. 637 to 641).
First, a layer of collagen gel (Base Layer) was prepared on a glass bottom dish.
Next, a ring-shaped aortic fragment (approximately 2 mm thick) was prepared for use in the experiment. The thoracic aorta was collected from the reporter mouse, and the outer membrane was removed from the collected thoracic aorta to prepare the aortic fragment.
Next, approximately two aortic fragments were placed on the dish perpendicular to the bottom of the culture dish, and the reconstituted collagen solution (0.3 mass % Cellmatrix typel-A, Nitta Gelatin) was layered on the dish. After the layering, the dish was left at 37° C. for 30 minutes, and the mixture (Base Layer, aorta fragments and reconstituted collagen solution) on the dish was gelatinized by leaving it at 37° C. for 30 minutes after the layering.
After the gelatinization, the control culture medium or the culture medium containing proteoglycans was added to the mixture. After the addition, the mixture to which the culture medium was added was incubated at 37° C. for 14 days. The group to which the control culture medium was added was designated as “control group” and the group to which the culture medium containing proteoglycans was added was designated as “PG-added group”. Cells elongated from the aortic fragments were checked by phase contrast microscopy. The total area of cells (indicated by arrows in
The results of the measurements are described below. When the total area of the control group was set at 100, the PG-added group was 555.2 (Student's t-test, p<0.001). Thus, the PG-added group showed significantly greater angiogenic activity than the control group.
The following samples were used in this experiment 6.
Using a model mouse with an injured sciatic nerve (sciatic nerve transected), we evaluated the ability of the unit containing VEGF, gel components (Matrigel) and proteoglycans to regenerate nerves and/or vascular regeneration capacity.
Experimental methods are described. First, 12-week-old male vascular reporter mice (Fltl-tdsRed BAC transgenic mouse) were created by transecting the sciatic nerves (sciatic nerves of the right and left limbs) toward the tail of the mouse (n=2). The right sciatic nerve of the model mice was treated with 10 mg/mL PG group, and the left sciatic nerve of the model mouse was treated with Control group.
Here, the following definitions are used.
Immediately after the treatment, the muscle layer and skin of the site of the treated model mice were sutured. After the suture, the model mice were reared normally for 5 days or 8 days. After the normal rearing, the model mice were dissected, and the treated areas were checked by the naked eye and stereomicroscope. After such confirmation, the sciatic nerve (about 5 mm) was collected from the model mice.
In this confirmation, compared to the treatment of control group (labeled as Control in
Using sciatic nerve injury model mice (sciatic nerve transected model mice), in vivo imaging was used to evaluate the ability to regenerate blood vessels by administering the unit containing a gel component (Matrigel) and proteoglycan. Evaluation using the imaging was performed using the IVIS Imaging System (PerkinElmer) including equipment.
Experimental methods are described. First, 9-week-old female vascular reporter mice (Fltl-tdsRed BAC transgenic mouse) were prepared by transecting the sciatic nerves (sciatic nerves of the right and left limbs) toward the tail of the mouse (n=1). The right sciatic nerve of the model mice was treated with 10 mg/mL PG group, and the left sciatic nerve of the model mice was treated with the Control group.
Here, the following definitions are used.
Immediately after the treatment, the muscle layer and skin of the site of the treated model mice were sutured. After said suture, the alfalfa-free fluorescence imaging feed (5V5M, PicoLabR SelectMouse 50IF/9F) was also used for 7 days of normal rearing of the model mice.
On the seventh day of the breeding, the AngioSense™ 750EX (PerkinElmer, NeV10011EX) was injected into the tail vein of the model mice (dose of 2 nmol/100 μL of AngioSense™ 750 EX per mouse). Normal rearing was continued for a certain period of time after the injection. The IVIS of the model mice were photographed at 18, 21, and 24 hours after the injection.
The conditions for such photography are shown below.
Immediately before the imaging, the model mice were given general anesthesia, and after the anesthesia, body hair was removed from the predetermined area of the model mice. After the hairs were removed, the images were taken. The general anesthesia was performed by placing the model mice in a suction anesthesia box with anesthesia circulating for a certain period of time. The removal was performed in the vicinity of the site where the above-mentioned treatment (PG group treatment or Control group treatment).
The results taken are shown in
In the evaluation shown in
Using a model mouse with an injured sciatic nerve (sciatic nerve transected), we checked whether administration of the unit containing a gel component (Matrigel) and proteoglycan causes angiogenesis and nerve regeneration by immunohistochemical staining.
Experimental methods are described. First, 12-week-old female vascular reporter mice (Flkl-GFP-BAC transgenic mice) were prepared by transecting the sciatic nerves (sciatic nerves of the right and left limbs) toward the tail of the mouse (n=1). The right sciatic nerve of the model mice was treated with 10 mg/mL PG group, and the left sciatic nerve of the model mice was treated with the Control group.
Here, the following definitions are used.
Immediately after the treatment, the muscle layer and skin of the site of the treated model mice were sutured. After said suture, the alfalfa-free fluorescence imaging feed (5V5M, PicoLabR SelectMouse 50IF/9F) was also used for 8 days of normal rearing of the model mice. After 8 days of normal rearing, the sciatic nerves of the right and left limbs of the model mice were collected. The right limb was the limb treated with the 10 mg/mL PG group, and the left limb was the limb treated with the Control group.
The sciatic nerve of the right limb and the sciatic nerve of the left limb that were collected were washed in PBS for 5 min. After the wash, the cells were fixed in 4% paraformaldehyde for 1 hour. After the fixation, the cells were replaced with a 30 mass % sucrose solution. After the replacement, the sciatic nerve of the right limb and the sciatic nerve of the left limb were embedded using an embedding agent for frozen tissue section preparation (Tissue Tech O.C.T. Compound, Sakura Finetech Japan). Samples were prepared. After the embedding, the embedded samples were placed at −80° C. for 3 hours. The frozen sections of the sciatic nerve of the right limb and the sciatic nerve of the left limb were prepared by this incubation. Cryostat HM525NX (Thermo Fisher Scientific) was used to cut the frozen sections to prepare sections at 100 μm thickness.
The 100 μm-thick sections were blocked with blocking buffer for 3 hours at room temperature (about 25° C.) After blocking, the sections were incubated with a reaction solution (primary antibody reaction solution) prepared by diluting a predetermined antibody with 10% Donkey Serum/PBS, and the primary antibody reaction was performed overnight (about 8 hours) at 4° C. After the primary antibody reaction, the antibody was intercepted using 0.3% TritonX-100/PBS (5 min at room temperature ×3 times). After the washing, the sections were diluted with 10% Donkey Serum/PBS (secondary antibody reaction solution) for 3 hours at room temperature. The secondary antibody reaction was performed for 3 hours at room temperature. After the secondary antibody reaction, the antibody was intercepted using 0.2% TritonX-100/PBS (5 min at room temperature xl time). After washing, the sections were sealed using the specified mounting agent. The sections after sealing were examined using a confocal microscope (Olympus Corporation: Confocal Laser Scanning Microscope FV3000) at a magnification of 4× to check whether the angiogenesis and nerve regeneration occurred.
The composition of the liquid used in the preparation of such sections described above is as follows.
The primary antibody reaction was performed with the primary antibody reaction solutions shown in the following Table 10.
The secondary antibody reaction was performed with the secondary antibody reaction solutions shown in Table 11 below.
The results of this experiment 9 are shown in
As shown in “VEGFR2-EGFP”, compared to the group of control group treatment (labeled “Control” in
Using a model mouse with an injured sciatic nerve (sciatic nerve transected), whether the administration of the unit containing a gel component (Matrigel) and proteoglycans improves motor function was evaluated by BBB scoring.
BBB scoring is one of the open field tests. BBB scoring is a method of evaluation that involves observing the behavior of subjects (in this experiment, the model mice treated with the following control group treatments, and model mice treated with the 10 mg/mL PG group treatment), when they are released into a space of about 40 to 60 cm square. Table 12 shows the BBB scoring for this experiment 10.
Experimental methods are described. First, 6-week-old female vascular reporter mice (Fltl-tdsRed BAC transgenic mice) were prepared. The sciatic nerves toward the tail of the two mice (sciatic nerves in the right and left limbs) were severed to create the model mice.
One model mouse (n=1) was given a control group treatment at the amputated site. The other model mouse (n=1) received 10 mg/mL PG group treatment at the transected site.
Here, the following definitions are used.
Immediately after the treatment, the muscle layer and skin of the site of the treated model mice were sutured. After the suture, the model mice were reared as usual and BBB scoring (counting of scores based on Table 12) was performed. The results are shown in Table 13. In Table 13, “2 hours” refers to the score at 2 hours after the suture, “1 day” refers to the score at 1 day after the suture, and “8 days” refers to the number of points obtained 8 days after the suture, “17 days” refers to the number of points obtained 17 days after the suture.
As shown in Table 13, compared to the group of Control group treatment, the group of PG group treatment showed an improvement in motor function (the hindlimb treated in this study always showed a vigorous kicking motion with the toes, and the direction of the toes was parallel to the trunk both at ground contact and at release) at “17 days”.
Using a model mouse with an injured sciatic nerve (sciatic nerve transected model mouse), in vivo imaging was used to evaluate the regenerative potential of blood vessels by administration of the unit containing gel components (Matrigel) and proteoglycans, and the like. Evaluation using the imaging was performed using the IVIS Imaging System (PerkinElmer) including equipment.
Experimental methods are described. First, 9-week-female vascular reporter mice (Fltl-tdsRed BAC transgenic mice) were prepared. The sciatic nerves toward the tail of the mice (sciatic nerves in the right and left limbs) were severed to create the model mice (n=1). The left sciatic nerve of the model mice was treated with the 10 mg/mL PG group, and the right sciatic nerve of the model mice was treated with the PG+SU4312 group.
Here, the following definitions are used.
Immediately after the treatment, the muscle layer and skin of the site of the treated model mice were sutured. After the suture, the alfalfa-free fluorescence imaging feed (5V5M, PicoLabR SelectMouse 50IF/9F) was also used for 7 days of normal rearing of the model mice.
On the seventh day of the breeding, the AngioSense™ 750EX (PerkinElmer, NeV10011EX) was injected into the tail vein of the model mice (dose of 2 nmol/100 μL of AngioSense 750 EX per mouse). Normal rearing was continued for a certain period of time after the injection. The IVIS of the model mice were photographed 24 hours after the injection.
The conditions for such photography are shown below.
Immediately before the imaging, the model mice were given general anesthesia, and after the anesthesia, body hair was removed from a predetermined area of the model mice. After the hairs were removed, the images were taken. The general anesthesia was performed by placing the model mice in a suction anesthesia box with anesthesia circulating for a certain period of time. The removal was performed in the vicinity of the site where the above-mentioned treatment (PG group treatment or Control group treatment).
The results of the imaging are shown in
In the group shown in
[Experiment 12: Confirmation of Aggrecan Expression, Etc. In Mouse Brain Tissue]
In order to understand how aggrecan (proteoglycan) functions in the process of angiogenesis in brain, we checked its expression. Aggrecan is a major structural proteoglycan present in the extracellular matrix of cartilage tissue. Its molecular weight is over 2,500 kDa, and it is composed of 100 to 150 glycosaminoglycan (GAG) chains attached to the core protein. Versican is a large proteoglycan distributed in a wide range of tissues. It has a complex structure with many N- and O-linked sugar chains. The core protein of versican has hyaluronic acid binding ability and is responsible for the organization and maintenance of the extracellular matrix.
The experimental methods for this experiment 12 are described. Brains from 15.5-day old mice , 17.5-day old mice, 1.5-month-old mice and 14.5-month-old mice were collected. The mice used in this experiment 10 were male vascular reporter mice (Fltl-tdsRed BAC transgenic mouse).
The brain samples were washed with PBS for 5 minutes. After the wash, cells were fixed in 4% paraformaldehyde for 1 hour. After fixation, the brains were embedded in a S% agarose gel. The encapsulated brains were cut using a vibratome, and 150-μm-thick sections were prepared. The sections were blocked in PBS solution containing 0.3% Triton and 10% Donkey serum for one day and night. After the blocking, the sections were subjected to a primary antibody reaction in a reaction solution containing the prescribed antibody (primary antibody reaction solution) for one day and night. After the primary antibody reaction, the sections were washed with PBS, and the washed sections were subjected to a secondary antibody reaction in a secondary antibody reaction solution for one day and night. After the secondary antibody reaction, the sections were washed with PBS, and the washed sections were sealed with the prescribed mounting agent.
The sections after such inclusion were examined using a confocal microscope (Olympus Corporation: Confocal Laser Scanning Microscope FV3000) at magnifications of 10× (
The results of this experiment 12 are shown in
In
In this 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.
To confirm that the cells cultured in the presence of FGF-2, EGF and proteoglycans are neural stem cells, the number of formed neurospheres (NS) were measured and the morphology of NS was observed.
The procedure (experimental procedure) for culturing neural stem cells in Experiment 13 is described below. A schematic diagram of this experimental procedure is shown in
The composition of the liquid culture medium for each group (control, culture medium 1, culture medium 2, and culture medium 3) is as follows. DMEM/HamF-12 (NACALAI TESQUE, INC., 08460-95) was used as the basic medium in all groups, and the composition of each component in 50 ml is shown below.
The ingredients contained in each of these groups (control, culture medium 1, culture medium 2, and culture medium 3) were as follows.
The results of this measurement are shown in
The results of this observation are shown in
Therefore, it was found that much more neural stem cells proliferate by the inclusion of FGF-2, EGF and proteoglycans.
By checking the transcriptional activity of Hes-1, a protein (transcription factor) that regulates the maintenance of undifferentiated state of neural stem cells and their differentiation into astrocytes, we confirmed whether the neural stem cells described above (cells cultured in the presence of FGF-2, EGF, and proteoglycans) were maintained in their undifferentiated state.
The procedure for this dual-luciferase assay is described below. First, neural stem cells were prepared. A schematic diagram of this experimental procedure is shown in
These samples were genetically transfected with Hes-1 cDNA and reporter gene (luciferase gene) using transfection reagent (Neon (registered trademark) Transfection System, Thermo Fisher Scientific). The Hes-1 cDNA and reporter gene (luciferase gene) were “pHes1(2.5 k)-luc (Plasmid #43806)” manufactured by Addgene. After this gene transfer, the cells were cultured in the liquid culture medium of each treatment group (control, culture medium 1, culture medium 2, culture medium 3, culture medium 4, culture medium 5, and culture medium 6) at 37° C. and 5% CO2 for 1 day. After 1 day of culture, luciferase substrate (Thermo Fisher Scientific, Neon™ Transfection System) was added to each sample, and the expression of luciferase in each sample was observed using a detection system (Promega Corporation, GloMax™ Discover Microplate Reader).
The composition of the liquid culture medium for each dose group (control, culture medium 1, culture medium 2, culture medium 3, culture medium 4, culture medium 5, and culture medium 6) used in this dual-luciferase assay is as follows. The composition of the control, culture medium 1, culture medium 2, and culture medium 3 is the same as the liquid culture medium used in Experiment 1 above. DMEM/HamF-12 (NACALAI TESQUE, INC., 08460-95) was used as the basic medium in all dose groups, and the composition of each component in 50 ml is shown below. In this culture, “Costar™ 24-well” from Corning Inc. was used.
(Culture Medium 1)
(Culture Medium 5)
The ingredients contained in each of these treatment groups (control, culture medium 1, culture medium 2, culture medium 3, culture medium 4, culture medium5 and culture medium6) were as follows.
The observation of the expression level of luciferase (transcriptional activity of Hes-1) in each well is described below. When the expression level of the control group was 100, the expression level of the group in culture medium 1 was 123.83, the expression level of the group in culture medium 2 was 131.24, the expression level of the group in culture medium 3 was 159.51, the expression level of the group in culture medium 4 was 113.85, the expression level of the group in culture medium 5 was 119.67, and the expression level of the group in culture medium 6 was 131.55. The higher the expression level of luciferase, the higher the transcriptional activity of Hes-1, and the more undifferentiated neural stem cells are maintained. Culture medium 6 is considered as a positive control, which maintains the undifferentiated potential of neural stem cells. However, compared to culture medium 6 (which does not contain proteoglycans but has 3-fold higher bFGF content compared to the control), culture medium 1, culture medium 2, and culture medium 3 groups (FGF-2, EGF and proteoglycans) maintained the undifferentiated nature of neural stem cells.
In a culture medium containing GAGs instead of proteoglycans, we checked whether cells cultured in this culture medium could form NSs. In this experiment, GAGs, unlike proteoglycans, do not have a core protein bound to them.
The procedure for this experiment 15 is described below. A schematic diagram of this experimental procedure is shown in
The composition of the liquid culture medium for each treatment group (control, culture medium 2, culture medium 7) is as follows. The composition of the control and culture medium 2 is the same as the liquid culture medium used in Experiment 13 above. DMEM/HamF-12 (NACALAI TESQUE, INC., 08460-95) was used as the basic culture medium in all dose groups, and the composition of each component in 50 ml is shown below.
The ingredients contained in each of these dose groups (control, culture medium 2, culture medium 7) were as follows.
The results of the measurement of the number of formed NSs is described below. The number of formed NSs in the control group was 100(%), and the percentage of
NSs formed in the control group was 58.8%. The number of NSs formed in the culture medium 2 group was 118.7%, and that in the culture medium 7 group was 58.8%. It was suggested that the structure of proteoglycans (the conjugate structure of the core protein and the GAG structure) promotes the formation of NS.
In neural stem cells grown in medium containing proteoglycans and other substances, we checked where the proteoglycans were localized.
This confirmation procedure is described below. A schematic diagram of this experimental procedure is shown in
First, neural stem cells were prepared. A female mouse on the 14th day of gestation was prepared using ICR mice manufactured by Japan SLC Co. Then, the mouse fetus present in the body of the mouse was removed, and cells dispersed from the cerebral cortex of the mouse fetus were prepared. In this dispersion, 0.05 w/v % trypsin (prepared by diluting a product of FUJIFILM Wako Pure Chemical Corporation 0.25 w/v % Trypsin-lmmol/L EDTA-4Na solution (containing phenol red) (product code: 201-16945, 209-16941))in PBS was used. The following medium groups (medium 2 and medium 8) were prepared using 180,000 of these dispersed cells. These cells were used separately, 60,000 cells per sample. A 6-well plate (Thermo Fisher Scientific Corporation, CAT #140675) in which the prescribed liquid medium is present). 6-well plates were inoculated with 2 ml of the prescribed liquid medium (the medium described in the relevant medium group) per well, and these cells were inoculated evenly into each well so that 10,000 cells were present. After this inoculation, the neural stem cells were cultured for 24 hours at 37° C., 5% CO2 to grow the neural stem cells for observation. One hour before collecting the neural stem cells to be used for observation, Hoechst 33342 (final concentration of 10 mg/mL, Hoechst™ 33342 imaging protocol (Thermo Fisher Scientific) was added and stained with the nuclei of neural stem cells. Observations were made using a fluorescence microscope (Olympus Corporation: Inverted Research Microscope IX81) at 20× magnification with the localization of proteoglycans in the formed cells in 6-well plates of each treatment group after 24 hours of culture was observed.
The composition of the liquid medium of each culture group (medium 2 and medium 8) used in this experiment 16 is as follows. Medium 2 has the same composition as the liquid medium used in Experiment 13 above. In all treatment groups, DMEM/Ham F-12 (NACALAI TESQUE, INC., 08460-95) was used as the basic medium, but in the following composition of each component in 50 ml.
The components contained in each of these culture media groups (culture media 2 and 8) were as follows.
The “fluorescently labeled proteoglycans” in medium 8 were prepared as follows: ATTO 488 NHS ester (Sigma-Aldrich) and proteoglycans were combined with the manufacturer's recommended method (product: ATTO 488 NHS ester (Sigma-Aldrich)) for 2 hours at room temperature. After this reaction, the fluorescently labeled proteoglycans were purified using Zebra Spin Desalting Column (Thermo Fisher Scientific) and recovered. The recovered fluorescently labeled proteoglycans were used in this experiment 16.
The results of the observations are shown in
The location of proteoglycans in neural stem cells grown in medium containing proteoglycans and other substances was also confirmed using confocal microscopy.
The procedure for this confirmation is described below. First, neural stem cells were prepared. A female mouse on the 14th day of gestation was prepared using ICR mice manufactured by Japan SLC Co. Then, the mouse fetus present in the body of the mouse was removed, and cells dispersed from the cerebral cortex of the mouse fetus were prepared. In this dispersion, 0.05 w/v % trypsin (prepared by diluting a product of FUJIFILM Wako Pure Chemical Corporation 0.25 w/v % Trypsin-1 mmol/L EDTA-4Na solution (containing phenol red) (product code: 201-16945, 209-16941))in PBS was used. The following medium group (medium 9) was prepared using 180,000 of these dispersed cells. These cells were used separately, 60,000 cells/each sample. The cells were inoculated into a 6-well plate (Thermo Fisher Scientific, Inc., CAT #140675) in which the prescribed liquid culture medium was present. 2 ml of the prescribed liquid culture medium was inoculated evenly into each well of the 6-well plate so that 10,000 cells of these cells were present per well. After the inoculation, the cells were cultured at 37° C., 5% CO2 for 24 hours to allow the neural stem cells to grow and to prepare them for observation. The neural stem cells used for observation were prepared. One hour before collecting the neural stem cells to be used for observation, Hoechst 33342 was used to stain the nuclei of the neural stem cells (final concentration 10 mg/mL, and Hoechst™ 33342 imaging protocol (Thermo Fisher Scientific)) and Cell mask (final concentration of 5mg/mL CellMask™ staining (Thermo Fisher Scientific) was added to stain and the cells were collected by centrifugation at 400 g for 3 min, followed by fixation in 4% paraformaldehyde for 10 min. The cells thus prepared were washed with PBS and mounted on glass slides with an encapsulant to prepare observation materials. Observations were made using a confocal microscope (Olympus Corporation: Confocal Laser Scanning Microscope FV3000) at 40× magnification to compare the localization of nuclei of neural stem cells , plasma membrane of neural stem cells and proteoglycans.
The composition of the liquid medium for each culture group (medium 9) used in this experiment 17 is as follows. Note that DMEM/Ham F-12 (NACALAI TESQUE, INC. 08460-95) was used, and in the following, the composition of each component in 5 ml is shown below.
The following ingredients were used in this medium 9.
The “fluorescently labeled proteoglycans” in medium 9 were prepared as follows: Atto 590 (Sigma-Aldrich, 70425) and proteoglycans were prepared by the method recommended by the manufacturer (product: Atto 590 (Sigma-Aldrich, 70425) for 2 hours at room temperature. After this reaction, Zeba Spin Desalting Column (Thermo Fisher Scientific) was used for purification, and the fluorescently labeled proteoglycans were recovered. The recovered fluorescently labeled proteoglycans were used in this experiment 5.
The results of the observation are shown in
While embodiments of the present invention (including examples) have been described above with reference to the drawings, the specific configuration of the present invention is not limited thereto, and even if there is a design change or the like within a range not departing from the gist of the present invention, it is included in the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-069686 | Apr 2020 | JP | national |
| 2020-085736 | May 2020 | JP | national |
| 2020-144278 | Aug 2020 | JP | national |
| 2021-013172 | Jan 2021 | JP | national |
This application is a National Phase of International Application No. PCT/JP2021/014496 filed Apr. 5, 2021, which claims priority based on Japanese Patent Applications No. 2020-069686 filed in Japan on Apr. 8, 2020, No. 2020-085736 filed on May 15, 2020, No. 2020-144278 filed on Aug. 28, 2020,and No. 2021-013172 filed on Jan. 29, 2021, the entire contents of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/014496 | 4/5/2021 | WO |
