Production method of extracellular vesicle population, and extracellular vesicle population

Abstract

The present invention provides a production method of an extracellular vesicle population containing extracellular vesicles on the membrane surface of which phosphatidylserine is low expressed, said method comprising: (1) a step for culturing mesenchymal stem cells in a medium containing one or more sulfuric acid compounds selected from the group consisting of a sulfated glycolipid, cholesterol sulfate and pharmaceutically acceptable salts thereof; and (2) a step for collecting an extracellular vesicle population contained in the medium after carrying out the above step (1). The present invention also provides an extracellular vesicle population containing extracellular vesicles on the membrane surface of which phosphatidylserine is low expressed.

Description

Method for producing extracellular vesicle population and extracellular vesicle population

[0001] The present invention relates to a population of extracellular vesicles containing extracellular vesicles with low expression of phosphatidylserine on the membrane surface, and a method for producing the same.

[0002] In recent years, attention has been focused on exosomes, a type of extracellular vesicles (EVs) secreted by cells. In particular, it has been reported that exosomes secreted by mesenchymal stem cells (MSCs) present in bone marrow, adipose tissue, and umbilical cord tissue exhibit therapeutic effects against various tissue injuries and diseases (Patent Documents 1 and 2 and Non-Patent Document 1).

[0003] To utilize exosome-containing extracellular vesicles as medicines, various technologies are currently under development, including those for promoting the secretion of extracellular vesicles produced by cells and for efficiently recovering the extracellular vesicles (see, for example, Patent Documents 3 and 4). It has been reported that stimulation of bone marrow-derived mesenchymal stem cells with sulfated glycolipid sulfatide promotes the secretion of exosomes, a type of extracellular vesicle (Non-Patent Document 2).

[0004] The membranes that make up EVs, such as exosomes, originate from the cell membranes that secrete them. The membranes of EVs contain a higher proportion of phosphatidylserine, a type of phospholipid, than cells, and it is exposed on the outside of the membrane (Non-Patent Document 3). On the other hand, in the cell membranes of living cells, phosphatidylserine is oriented on the inside due to the action of an enzyme called flippase. When a cell undergoes apoptosis, phosphatidylserine becomes oriented on the outside of the cell membrane, which is recognized by microglia and macrophages and phagocytoses the apoptotic cell (e.g., Non-Patent Documents 4 and 5). Therefore, EVs with phosphatidylserine exposed on the outside of the membrane may be captured and phagocytosed by microglia and macrophages that recognize phosphatidylserine, just like apoptotic cells, before reaching the site where the therapeutic effect is desired. This may reduce the therapeutic effect of administering EVs.

[0005] Currently, no EVs such as exosomes with reduced phosphatidylserine content or methods for producing them are known.

[0006] Japanese Patent Application Laid-Open No. 2020-174573 Japanese Patent Application Laid-Open No. 2020-522996 Japanese Patent Application Laid-Open No. 2021-122206 Japanese Patent Application Laid-Open No. 2021-122205

[0007] Katsuda T. and Ochiya T., Molecular signatures of mesenchymal stem cell-derived extracellular vesicle-mediated tissue repair, Stem Cell Research &Therapy, 6: 212, 2015. Yamashita T., Tsuru E., Wang Feifei, Baba N., Shen Yuan, Tsuda M., Maeda N., Honke K., Sagara Y., Stimulation of exosome secretion from mesenchymal stem cells by sulfated glycolipids. 16th Annual Meeting of the Japanese Society for Regenerative Medicine, March 7-9, 2017, Sendai. Trajkovic K. et al., Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 319(5867) : 1244-7(2008). Moseman EA, et al., T cell engagement of cross-presenting microglia protects the brain from a nasal virus infection, Sci Immunol. 2020 Jun 5;5(48):eabb1817.Herbert RP, et al., Cytokines and olfactory bulb microglia in response to bacterial challenge in the compromised primary olfactory pathway, J Neuroinflammation. 2012 May 29;9:109.Wang F., Yamashita T. et al., The therapeutic potential of human umbilical cord blood transplantation for neonatal hypoxic-ischemic brain injury and ischemic stroke, Acta Med. Okayama, 66 (6), 429-434, 2012Wang F., Yamashita T. et al., Syngeneic transplantation of newborn splenocytes in a murine model of neonatal ischemia-reperfusion brain injury, J. Maternal-Fetal & Neonatal Med., 28, 2015.

[0008] The object of the present invention is to provide extracellular vesicles that can reach a desired treatment site and exert a therapeutic effect, and a method for producing the same.

[0009] The present inventors discovered that stimulating mesenchymal stem cells with sulfated compounds (e.g., sulfated glycolipids or cholesterol sulfate) not only promoted the secretion of EVs, but also produced EVs that contained little phosphatidylserine. Furthermore, they discovered that the EVs thus obtained exhibited significant therapeutic effects in HIE model mice compared to EVs released in the absence of sulfated compounds. Based on these new findings, the present invention was completed.

[0010] That is, the present invention includes the following inventions. [1] A method for producing a population of extracellular vesicles containing extracellular vesicles with low expression of phosphatidylserine on the membrane surface (low-PS extracellular vesicles), the method comprising: (1) a step of culturing animal cells in a medium containing one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharmaceutically acceptable salts thereof, and (2) a step of recovering the population of extracellular vesicles contained in the medium after performing the step (1). [2] The method according to item 1, wherein the sulfated glycolipid is one or more selected from the group consisting of sulfatides and seminolipids. [3] The method according to item 1 or 2, wherein the animal cells are cells that highly express Growth Differentiation Factor-15 (GDF-15). [4] The method according to Item 3, wherein the cells highly expressing GDF-15 are mesenchymal cells, female germline cells, bone marrow-derived cells, endothelial cells, hepatic cells, renal cancer-derived cells, or neuroblastoma-derived cells. [5] The method according to Item 3, wherein the cells highly expressing GDF-15 are mesenchymal stem cells. [6] The method according to Item 5, wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord, dental pulp, synovium, placenta, and / or fetal membrane, and / or mesenchymal stem cells obtained by inducing differentiation of pluripotent stem cells. [7] The method according to Item 5, wherein the mesenchymal stem cells are mesenchymal stem cells derived from umbilical cord. [8] The method according to any one of Items 1 to 7, wherein the phosphatidylserine content in the PS-low extracellular vesicles is 0.5-fold or less compared to that in extracellular vesicles secreted from the animal cells cultured in the absence of the sulfate compound. [9] The method according to any one of items 1 to 8, wherein the extracellular vesicle population comprises extracellular vesicles with a high expression of phosphatidylserine on the membrane surface (PS-rich extracellular vesicles).

[10] The method according to any one of items 1 to 9, wherein the extracellular vesicle population is used for the treatment of a nervous system disease.

[11] The method according to item 10, wherein the nervous system disease is ischemic encephalopathy or traumatic brain injury.

[12] The method according to any one of items 1 to 11, wherein the extracellular vesicle population comprises extracellular vesicles having a high content of microRNA selected from the group consisting of miR-17 and miR-106.

[13] The method according to any one of Items 1 to 12, wherein the extracellular vesicle population comprises extracellular vesicles with a high content of GDF-15.

[14] An extracellular vesicle population comprising extracellular vesicles with low expression of phosphatidylserine on the membrane surface (low PS extracellular vesicles), obtained by the method according to any one of Items 1 to 13.

[15] An extracellular vesicle population comprising extracellular vesicles derived from animal cells with low expression of phosphatidylserine on the membrane surface (low PS extracellular vesicles).

[16] The extracellular vesicle population according to Item 15, wherein the extracellular vesicles are derived from cells with a high expression of Growth Differentiation Factor-15 (GDF-15).

[17] The extracellular vesicle population according to Item 16, wherein the cells with a high expression of GDF-15 are mesenchymal stem cells, female germline cells, bone marrow-derived cells, renal cancer-derived cells, or neuroblastoma-derived cells.

[18] The extracellular vesicle population according to Item 16, wherein the cells highly expressing GDF-15 are mesenchymal stem cells.

[19] The extracellular vesicle population according to Item 18, wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord, dental pulp, synovium, placenta, and / or fetal membrane, and / or mesenchymal stem cells obtained by inducing differentiation from pluripotent stem cells.

[20] The extracellular vesicle population according to Item 18, wherein the mesenchymal stem cells are mesenchymal stem cells derived from umbilical cord.

[21] The extracellular vesicle population according to any one of Items 15 to 20, wherein the phosphatidylserine content in the low PS extracellular vesicles is 0.5-fold or less compared to that in extracellular vesicles secreted from the animal cells cultured in the absence of one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharmaceutically acceptable salts thereof.

[22] The extracellular vesicle population according to any one of Items 15 to 21, wherein the extracellular vesicle population comprises extracellular vesicles with high expression of phosphatidylserine on the membrane surface (PS-rich extracellular vesicles).

[23] The extracellular vesicle population according to any one of Items 15 to 22, wherein the extracellular vesicle population is used for the treatment of a nervous system disease.

[24] The extracellular vesicle population according to Item 23, wherein the nervous system disease is ischemic encephalopathy or traumatic brain injury.

[25] The extracellular vesicle population according to any one of Items 15 to 24, wherein the extracellular vesicle population comprises extracellular vesicles rich in microRNA selected from the group consisting of miR-17 and miR-106.

[26] The extracellular vesicle population according to any one of Items 15 to 25, wherein the extracellular vesicle population comprises extracellular vesicles rich in GDF-15.

[27] A method for treating or preventing a nervous system disease in a subject in need thereof, comprising administering to the subject the extracellular vesicle population according to any one of Items 15 to 26.

[0011] The present invention makes it possible to provide extracellular vesicles that, unlike previously known EVs, have low expression of phosphatidylserine on their membrane surface. This makes it possible to provide extracellular vesicles that can reach desired sites and exert their effects without being captured by phagocytes such as microglia and macrophages. Furthermore, the present invention makes it possible to provide a population of extracellular vesicles that can cure nervous system diseases that have been difficult to treat until now, such as intractable central nervous system disorders.

[0012] Figure 1 shows the results of comparing the effects of the presence or absence of sulfatide on the amount of extracellular vesicles (EVs) secreted from human umbilical cord-derived mesenchymal stem cells. Figure 2 shows the results of comparing the effects of different amounts of added sulfatide on the amount of extracellular vesicles (EVs) secreted from human umbilical cord-derived mesenchymal stem cells. Figure 3 shows the results of comparing the effects of different added sulfate compounds on the amount of extracellular vesicles (EVs) secreted from human umbilical cord-derived mesenchymal stem cells. Figure 4 shows the results of comparing the expression of marker proteins in extracellular vesicles (EVs) obtained by culturing human umbilical cord-derived mesenchymal stem cells with or without sulfatide and purifying the culture supernatant using ultracentrifugation or Mag Capture™ Exosome Isolation Kit PS. Figure 5 shows the results of comparing phosphatidylserine (PS) on the surface of extracellular vesicles (EVs) obtained by culturing human umbilical cord-derived mesenchymal stem cells with or without sulfatide and purifying the culture supernatant using ultracentrifugation or the Mag Capture™ Exosome Isolation Kit PS. The PS was detected by ELISA using Annexin V. Figure 6 shows fluorescently stained images of tissue sections from the coronal surface of the lateral ventricle of HIE model mice that received tail vein administration of extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells supplemented with sulfatide. Green: GFAP (a marker for the damaged area (glial scar)); Red: pKH26-labeled EVs. Figure 7 shows a fluorescent staining image of a tissue section from the coronal surface of the hippocampus of an HIE model mouse that received tail vein administration of extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells supplemented with sulfatide. Green: GFAP (a marker for damage sites (glial scars)); Red: pKH26-labeled EVs). Figure 8 shows a fluorescent staining image of a tissue section from the coronal surface of the lateral ventricle of a normal mouse that received tail vein administration of extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells supplemented with sulfatide. Green: GFAP (a marker for damage sites (glial scars)); Red: pKH26-labeled EVs. Figure 9 shows a fluorescent staining image of a tissue section from the coronal surface of the lateral ventricle of an HIE model mouse that received intranasal administration of extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells supplemented with sulfatide. Green: GFAP (a marker for the damaged area (glial scar)), red: pKH26-labeled EVs.Figure 10 shows a fluorescent staining image of a tissue section from the coronal surface of the lateral ventricle of an HIE model mouse that was intranasally administered extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells without the addition of sulfatide. Green: GFAP (a marker for the damaged area (glial scar)); Red: pKH26-labeled EVs. Figure 11 is a schematic diagram showing the changes in the properties of extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells with or without sulfatide. Figure 12 shows the motor function recovery effect (rotarod test) of extracellular vesicles (EVs) in normal mice and HIE model mice. Figure 13 shows the neurodifferentiation-promoting effect of extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells cultured in medium with or without sulfatide on mouse neural stem cells. Figure 14 shows the results of quantifying microRNAs contained in extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells cultured in a medium with or without sulfatide. Figure 15 shows the effect of adding extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells cultured in a medium with or without sulfatide on the expression of miR-17 / 106a target molecules in neuroblastoma cells SHSY5Y. Figure 16 shows the results of detecting GDF-15 protein contained in extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells cultured in a medium with or without sulfatide. Figure 17 shows the relationship between the amount of sulfatide added to the medium and the amount of GDF-15 contained in extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells. Figure 18 shows the relationship between the culture time after the addition of sulfatide to the medium and the amount of GDF-15 contained in extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells. Figure 19 shows the results of comparing the effects of different added sulfate compounds on the amount of GDF-15 contained in extracellular vesicles (EVs) from human umbilical cord-derived mesenchymal stem cells. Figure 20 shows the results of comparing the effects of the sulfated glycolipid seminolipid on the amount of GDF-15 contained in extracellular vesicles (EVs) from human umbilical cord-derived mesenchymal stem cells. Figure 21 shows the results of a search using a database ("THE HUMAN PROTEIN ATLAS") for cells highly expressing GDF-15.Figure 22 shows the results of examining the effect of adding sulfatide to the culture medium on the amount of GDF-15 contained in extracellular vesicles (EVs) obtained from K562 cells. Figure 23 shows the results of examining the effect of adding sulfatide to the culture medium on the amount of GDF-15 contained in extracellular vesicles (EVs) obtained from multiple cancer cell lines. Figure 24 shows the results of comparing the amount of GDF-15 contained in extracellular vesicles (EVs) obtained from each cell type.

[0013] Hereinafter, embodiments for carrying out the present invention will be described, but the technical scope of the present invention is not limited to the following embodiments. Note that the prior art documents cited in this specification are also incorporated herein by reference in their entirety.

[0014] In one embodiment, the present invention provides a method for producing an extracellular vesicle population comprising extracellular vesicles with low expression of phosphatidylserine on the membrane surface (low-PS extracellular vesicles), the method comprising: (1) a step of culturing mesenchymal stem cells in a medium containing one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharmaceutically acceptable salts thereof; and (2) a step of recovering the extracellular vesicle population contained in the medium after carrying out the step (1).

[0015] As used herein, "extracellular vesicles (EVs)" refer to particles covered with a lipid bilayer membrane that are secreted from cells, and include, for example, exosomes, microvesicles (MVs), apoptotic bodies, and the like.

[0016] In addition, as used herein, the term "extracellular vesicle population" refers to a population of extracellular vesicles containing multiple extracellular vesicles, and is used to distinguish it from individual extracellular vesicles.

[0017] Exosomes are small vesicles of approximately 20 to 200 nm derived from the endocytic pathway. Their components are known to include proteins, nucleic acids (mRNA, miRNA, non-coding RNA, etc.), and may have functions such as mediating intercellular communication. Because the mechanisms of exosome production and secretion remain largely unknown, it is difficult to strictly distinguish them from microvesicles (MVs). However, they can be roughly identified using commonly known exosome marker molecules, such as Alix, Tsg101, tetraspanins (e.g., CD81, CD63, CD9), flotillin, and phosphatidylserine. Exosomes may be identified using antibodies or binding proteins against these marker molecules. The components of exosomes are summarized in ExoCarta (http: / / www.exocarta.org / ), and the presence or absence of the components listed therein may be used as an indicator for exosome identification.

[0018] Microvesicles (MVs) are vesicles of about 50 to about 1000 nm derived from the cytoplasmic membrane, and are known to contain proteins, nucleic acids (mRNA, miRNA, non-coating RNA, etc.), etc., and may have functions such as controlling intercellular communication. Examples of marker molecules for microvesicles include integrin, selectin, CD40, CD154, etc., and microvesicles (MVs) can be identified by using antibodies or binding proteins against these marker molecules.

[0019] Apoptotic bodies are vesicles of approximately 500 to 2000 nm derived from the cytoplasmic membrane, and are known to contain fragmented nuclei, organelles, etc. as components thereof, and may have the function of inducing phagocytosis, etc. Marker molecules for apoptotic bodies include, for example, Annexin V and phosphatidylserine, and apoptotic bodies can be identified by using antibodies or binding proteins against these marker molecules.

[0020] In one embodiment, the extracellular vesicles included in the present invention preferably include exosomes. Generally, the distinction between types of extracellular vesicles (exosomes, microvesicles, apoptotic bodies, etc.) has not necessarily been scientifically clarified, and it is technically difficult to selectively isolate specific types of extracellular vesicles. Therefore, regardless of the method used to isolate extracellular vesicles, they will usually contain exosomes. Therefore, in the practice of the present invention, the modes and conditions for isolating extracellular vesicles are not limited to those that strictly distinguish between types of extracellular vesicles and target specific ones.

[0021] Through extensive research, the present inventors have found that culturing animal cells in a medium containing one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharmaceutically acceptable salts thereof increases extracellular vesicle production compared to conventional culture methods (e.g., a method of culturing animal cells in a medium that does not contain one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharmaceutically acceptable salts thereof). The present inventors have also demonstrated that the extracellular vesicle population provided by the present invention contains extracellular vesicles with low levels of phosphatidylserine on the membrane surface compared to conventional extracellular vesicles (e.g., extracellular vesicles produced from animal cells cultured in a medium that does not contain one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharmaceutically acceptable salts thereof). Furthermore, the present inventors have found that the extracellular vesicle population provided by the present invention, which contains extracellular vesicles with low levels of phosphatidylserine on the membrane surface, can exhibit favorable therapeutic effects when used to treat nervous system diseases.

[0022] In one embodiment, the extracellular vesicle population provided by the present invention is characterized by comprising extracellular vesicles with low expression of phosphatidylserine on the membrane surface.

[0023] In one embodiment, the "extracellular vesicles in which phosphatidylserine is expressed at a low level on the membrane surface" (hereinafter may be abbreviated as "low-PS extracellular vesicles") provided by the present invention refer to extracellular vesicles in which the amount of phosphatidylserine expressed at a low level on the membrane surface is lower than that of extracellular vesicles secreted from animal cells under conventional culture conditions (e.g., in the absence of one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharmaceutically acceptable salts thereof), and may be extracellular vesicles in which the amount of phosphatidylserine expressed is preferably 0.5-fold or less, for example, 0.25-fold or less, 0.1-fold or less, 0.05-fold or less, or 0.01-fold or less. The amount of phosphatidylserine expressed may be determined appropriately using a known method (e.g., ELISA, Western blotting, chromatography, etc.), and is not particularly limited. In this specification, the "expression level" of a substance may be referred to as the "content" of the substance, and may refer to, for example, a direct measurement of the content of the substance, or to a calculation of the content (or expression level) of the substance in a sample to be measured by comparison with a standard sample containing a known amount of the substance using any detection method. Alternatively, the expression level (content) may be a relative amount to a comparator, for example, a relative expression level (content) derived by comparison with the "expression level" (content) of the substance of interest contained in extracellular vesicles secreted from animal cells under conventional culture conditions (e.g., in the absence of one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharmaceutically acceptable salts thereof).

[0024] In one embodiment, the extracellular vesicle population provided by the present invention may comprise extracellular vesicles with high levels of phosphatidylserine on the membrane surface (high-PS extracellular vesicles). Furthermore, in one embodiment, the extracellular vesicle population provided by the present invention may comprise extracellular vesicles with low levels of phosphatidylserine on the membrane surface and extracellular vesicles with high levels of phosphatidylserine on the membrane surface (high-PS extracellular vesicles). Therefore, compared to extracellular vesicles secreted from animal cells under conventional culture conditions (e.g., in the absence of one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharmaceutically acceptable salts thereof), the extracellular vesicle population provided by the present invention has a skewed distribution of phosphatidylserine content on the membrane surface, and may comprise extracellular vesicles with low levels of phosphatidylserine (or no phosphatidylserine) on the membrane surface and extracellular vesicles with high levels of phosphatidylserine on the membrane surface.

[0025] "Extracellular vesicles in which phosphatidylserine is highly expressed on the membrane surface" (which may be abbreviated as "PS-rich extracellular vesicles" in this specification) refer to extracellular vesicles in which the amount of phosphatidylserine expressed on the membrane surface is higher than that of extracellular vesicles secreted from animal cells under conventional culture conditions (e.g., in the absence of one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharmaceutically acceptable salts thereof), and may be extracellular vesicles that preferably contain 1.5 times or more, for example, 2 times or more, 3 times or more, or 4 times or more, of phosphatidylserine.

[0026] In one embodiment, the extracellular vesicle population provided by the present invention contains extracellular vesicles enriched in microRNAs selected from the group consisting of miR-17 and miR-106. In another embodiment, the extracellular vesicle population provided by the present invention can be used in the treatment of nervous system diseases.

[0027] In one embodiment, animal cells applicable to the present invention are preferably, for example, cells that highly express Growth Differentiation Factor-15 (GDF-15). Cells that highly express GDF-15 can be identified using known databases, such as "THE HUMAN PROTEIN ATLAS" (URL: https: / / www.proteinatlas.org / ). Here, a method of identification using "THE HUMAN PROTEIN ATLAS" is exemplified, but such cells may also be identified using other databases (e.g., the National Library of Medicine (NCBI) provided by the U.S. National Institutes of Health). When searching for cells highly expressing GDF-15 using "THE HUMAN PROTEIN ATLAS," "highly GDF-15 expressing cells" may be identified based on the normalized expression (nTPM) value registered in the database. For example, tissues with an nTPM of 50 or more (e.g., 100 or more, or 150 or more) or cells or cell lines (e.g., cancer cell lines) derived from such tissues may be selected and used. Examples of such cells include female germline cells (e.g., BEWO cells), bone marrow-derived cells (e.g., K562 cells), mesenchymal cells (e.g., HHSteC cells), endothelial cells (e.g., TIME cells, HUVEC cells), and hepatic cells (e.g., HepG2) (see, for example, FIG. 21 ). Alternatively, the cells may be derived from kidney cancer (e.g., KMRC-20) or neuroblastoma (e.g., SHSY-5Y) (see, for example, Figure 23). Mesenchymal stem cells are particularly preferred as animal cells applicable to the present invention.

[0028] As used herein, "mesenchymal stem cells" refer to undifferentiated cells that have the ability to differentiate into various mesenchymal cells, such as adipocytes, chondrocytes, osteocytes, myoblasts, fibroblasts, stromal cells, and / or tenocytes, and that also have the ability to self-renew. The International Society for Cellular Therapy (ISCT) proposes the following three minimum criteria for defining mesenchymal stem cells: (1) the ability to be cultured by adhering to plastic under standard culture conditions, (2) immunological characteristics that are positive for CD105, CD73, and CD90, and negative for CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR, and (3) the ability to differentiate into osteoblasts, adipocytes, and chondroblasts in an in vitro differentiation system; however, the present invention is not limited to these criteria. Other positive markers that indicate mesenchymal stem cells include CD29, CD44, CD106, and STRO-1. In the present invention, the term "mesenchymal stem cells" is to be interpreted in the broadest possible sense.

[0029] Mesenchymal stem cells are cells that are isolated in vivo from tissues such as bone marrow, adipose tissue, umbilical cord, dental pulp, synovial membrane, placenta, and fetal membrane (e.g., amnion), and can be isolated using known methods.

[0030] For example, bone marrow-derived mesenchymal stem cells are prepared by separating blood cells from bone marrow fluid by density gradient centrifugation, seeding them on plastic culture dishes, and culturing them at 37°C and 5% CO ２ By culturing them in this environment, they can be isolated as adherent cells.

[0031] Adipose tissue-derived mesenchymal stem cells (adipose tissue-derived stem cells) are prepared by mincing collected adipose tissue, digesting the tissue with collagenase type II at 37°C for 1 hour, adding medium, and centrifuging. The precipitated cells are then washed with basal medium, filtered through a mesh such as a cell strainer, and seeded onto a plastic culture dish. The cells are then cultured at 37°C and 5% CO. ２ By culturing them in this environment, they can be isolated as adherent cells.

[0032] For example, umbilical cord-derived mesenchymal stem cells can be isolated by collecting umbilical cord tissue, finely chopping it, placing it on a petri dish, immersing it in a culture medium containing 10% bovine serum albumin, and culturing it for 10 to 20 days, and then recovering the proliferated cells.

[0033] The method for isolating mesenchymal stem cells derived from other tissues is not limited and may be any known method. Furthermore, the purity may be increased by using known mesenchymal stem cell markers, for example, by FACS or magnetic bead methods.

[0034] The mesenchymal stem cells may be mesenchymal stem cells obtained by inducing differentiation from pluripotent stem cells. As used herein, pluripotent stem cells refer to cells that have the ability to self-renew and multi-differentiation, and have the ability (pluripotency) to form all the cells that make up the body. Self-renewal ability refers to the ability of one cell to produce two undifferentiated cells identical to itself. Pluripotent stem cells used in the present invention include embryonic stem cells (ES cells), embryonic carcinoma cells (EC cells), trophoblast stem cells (TS cells), shrimp stem cells (EpiS cells), embryonic germ cells (EG cells), multipotent germ cells (mGS cells), and induced pluripotent stem cells (iPS cells). Mesenchymal stem cells induced from pluripotent stem cells using known methods (e.g., JP 2012-120486 A, Fukuta M., et al., "Derivation of mesenchymal stromal cells from pluripotent stem cells through a neural crest lineage using small molecule compounds with defined media," PLOS ONE, 2014; 9(12): e112291, etc.) may also be used.

[0035] The ability of mesenchymal stem cells to be induced to differentiate can be confirmed by known methods. For example, to confirm the induction of differentiation from mesenchymal stem cells into adipocytes, the cells can be cultured in a medium containing insulin and dexamethasone, and the confirmation can be made by Oil Red O staining. For example, to induce differentiation from mesenchymal stem cells into bone cells, the cells can be cultured in a medium containing ascorbic acid, β-glycerophosphate, and dexamethasone, and the confirmation can be made by alkaline phosphatase staining. For example, to induce muscle differentiation from mesenchymal stem cells, the cells can be cultured in a medium containing horse serum, and the appearance of fused cells specific to muscle cells can be confirmed. Other methods for inducing differentiation from mesenchymal stem cells can be used without particular limitation. The presence or absence of differentiation induction can also be confirmed by known methods, for example, by detecting genes or proteins that are expressed only after differentiation induction using real-time PCR, flow cytometry, or the like.

[0036] The origin of the mesenchymal stem cells used in the present invention is not limited, but from the viewpoints of ease of acquisition and therapeutic effect on nervous system diseases, they may be mesenchymal stem cells derived from bone marrow, adipose tissue, or umbilical cord, and mesenchymal stem cells derived from umbilical cord are preferred.

[0037] Examples of animal cells used in the present invention include mammals such as humans, rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, goats, monkeys, chimpanzees, and immunodeficient animals thereof, as well as birds, reptiles, amphibians, fish, and insects. When the present invention is used to treat humans, it is preferable to use mesenchymal stem cells derived from humans; when the present invention is used to treat pigs, it is preferable to use mesenchymal stem cells derived from pigs; when the present invention is used to treat monkeys, it is preferable to use mesenchymal stem cells derived from monkeys; and when the present invention is used to treat chimpanzees, it is preferable to use mesenchymal stem cells derived from chimpanzees. Furthermore, when treating humans, cells may be collected from the patient themselves (autologous cells), cells collected from other people (allogeneic cells), or commercially available cell lines.

[0038] In the present invention, mesenchymal stem cells refer to any cell population containing mesenchymal stem cells, in which at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98%, or 99% of the cell population is mesenchymal stem cells.

[0039] The medium used in step (1) of the present invention is not particularly limited as long as it is a medium capable of culturing animal cells, and may be prepared, for example, by adding serum to a basal medium and / or one or more serum substitutes such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. These media may further contain, as necessary, substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc.

[0040] Examples of basal media that can be used for animal cells include, but are not limited to, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, MCDB201 medium, and mixed media thereof.

[0041] Examples of serum to be added to the medium include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, porcine serum, sheep serum, rabbit serum, rat serum, etc. When serum is used, it may be added at 5 v / v % to 15 v / v %, preferably 10 v / v %, to the basal medium.

[0042] Examples of fatty acids that can be added to the medium include, but are not limited to, linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoylic acid, palmitic acid, and stearic acid. Examples of lipids that can be added to the medium include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Examples of amino acids that can be added to the medium include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, and L-glycine. Examples of proteins that can be added to the medium include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of polysaccharides that can be added to the culture medium include, but are not limited to, glycosaminoglycans, and particular examples of glycosaminoglycans include hyaluronic acid and heparan sulfate. Examples of growth factors that can be added to the culture medium include, but are not limited to, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), epidermal growth factor (EGF), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF).

[0043] The composition containing the extracellular vesicle population obtained in the present invention may be prepared using a xeno-free medium that does not contain xenogeneic components such as serum. Such media are available as pre-prepared media for mesenchymal stem cells (stromal cells) from, for example, PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning, and Rohto.

[0044] The present invention may use "one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharmaceutically acceptable salts thereof." Examples of sulfated glycolipids that may be used include sulfatide, seminolipid, and pharmaceutically acceptable salts thereof.

[0045] The pharmaceutically acceptable salts of sulfated glycolipids or cholesterol sulfate used in the present invention may be extracted from natural products or synthesized by known methods. For example, those listed in the 17th edition of the Japanese Pharmacopoeia can be used. Examples of pharmaceutically acceptable salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, tartrate, citrate, fumarate, maleate, toluenesulfonate, and methanesulfonate; metal salts such as sodium salt, potassium salt, calcium salt, and aluminum salt; and salts with bases such as triethylamine salt, guanidine salt, ammonium salt, hydrazine salt, quinine salt, and cinchonine salt.

[0046] The extracellular vesicle population provided by the present invention may be provided as a composition (e.g., a pharmaceutical composition) containing the above-mentioned extracellular vesicle population as well as a pharmacologically acceptable carrier, diluent, or excipient. The composition may be provided in a dosage form suitable for topical administration (cutaneous, inhalation, enema, eye drop, ear drop, nasal, vaginal, etc.), enteral administration (oral, tube feeding, intravenous injection, etc.), or parenteral administration (intravenous, intraarterial, transdermal, intramuscular injection, etc.), but the administration route is not limited thereto. The extracellular vesicle population provided by the present invention includes extracellular vesicles with low expression of phosphatidylserine on the membrane surface. Therefore, when administered nasally, for example, the extracellular vesicle population can reach the desired site (e.g., a damaged area of ​​the brain) without being trapped in the olfactory bulb where microglia that target phosphatidylserine accumulate.

[0047] The compositions provided by the present invention can be formulated in a dosage form suitable for the route of administration. For oral administration, capsules, liquids, etc. can be designed, while for parenteral administration, injections, infusion preparations, etc. can be designed. These formulations may also contain various pharmaceutical adjuvants, i.e., carriers and other auxiliary agents, such as stabilizers, preservatives, soothing agents, flavoring agents, corrigents, fragrances, emulsifiers, fillers, pH adjusters, etc., within the range that does not impair the effects of the compositions of the present invention.

[0048] The method for collecting and / or purifying the extracellular vesicle population may be any known method, such as ultracentrifugation, density gradient separation, ultrafiltration, immunoprecipitation, chromatography, or the like, or the collection and / or purification may be performed using various microparticle separation kits. Furthermore, in one embodiment, when it is desired to increase the concentration of extracellular vesicles with low expression of phosphatidylserine on the membrane surface (low-PS extracellular vesicles) in the extracellular vesicle population provided by the present invention, they may be enriched by a method of recognizing and isolating phosphatidylserine, for example, a method using magnetic beads onto which Tim4 (T-cell immunoglobulin domain and mucin domain-containing protein 4), which binds in a calcium ion-dependent manner, is immobilized (e.g., MagCapture™ Exosome Isolation Kit PS (Fujifilm Wako Pure Chemical Industries, Ltd., Japan)). Alternatively, for example, they may be enriched using magnetic beads onto which an anti-tetraspanin antibody (e.g., an anti-CD9 antibody, an anti-CD81 antibody, or an anti-CD63 antibody) is immobilized, and this can be carried out using an appropriate known technique.

[0049] In one embodiment, the extracellular vesicle population provided by the present invention contains extracellular vesicles that are rich in microRNAs selected from the group consisting of miR-17 and miR-106. The nucleic acid sequences of "miR-17" and "miR-106" can be identified using known databases (e.g., NCBI, HGNC, etc.). Furthermore, "miR-17" or "miR-106" contained in extracellular vesicles that can be applied to the present invention includes their orthologs (e.g., miR-106a and miR-106b, etc.).

[0050] MicroRNA (miRNA) generally refers to single-stranded non-coding RNA of approximately 21 to 25 bases that does not encode proteins, and it suppresses the expression of complementary or partially complementary messenger RNA (mRNA). One type of miRNA is said to be involved in regulating the expression of multiple genes. The gene expression regulation mechanism involving this miRNA is related to various biological phenomena, vital functions, diseases, etc.

[0051] As used herein, "extracellular vesicles rich in microRNA selected from the group consisting of miR-17 and miR-106" refers to extracellular vesicles having a high content of each microRNA compared to extracellular vesicles secreted from mesenchymal stem cells under conventional culture conditions (i.e., in the absence of sulfate compounds), and preferably refers to extracellular vesicles containing 1.5 times or more, for example, 2 times or more, or 3 times or more of the microRNA. The content of microRNA may be determined appropriately using known methods and is not particularly limited.

[0052] In one embodiment, the extracellular vesicle population provided by the present invention may include extracellular vesicles rich in Growth Differentiation Factor (GDF) family proteins, for example, GDF proteins selected from the group consisting of GDF-1, GDF-2, GDF-3, GDF-5, GDF-9, GDF-11, and GDF-15, particularly GDF-15.

[0053] GDF-15 is a member of the transforming growth factor β (TGF-β) superfamily, and is known to be secreted from macrophages, adipocytes, cardiac myocytes, vascular endothelial cells, vascular smooth muscle cells, and the like. In recent years, GDF-15 has been reported to be involved in neural differentiation and neural stem cell neogenesis and to be associated with therapeutic effects against Alzheimer's disease (see, for example, Dong Hyun Kim et al. STEM CELLS AND DEVELOPMENT Volume 24, Number 20, 2015; Yuelin Zhang et al. Scientific Reports, 5: 11235, 2015; Wen-Ping Xiong et al. Brain Research Bulletin 177 (2021) 92-102). It is known that when GDF-15 mRNA is transcribed and translated into protein, it becomes approximately 34 kDa pro-GDF-15 (immature form), which is then cleaved by PCSK-3, PCSK-5, or PCSK-6 of the proprotein convertase, subtilisin / kexin-type (PCSK) family to become approximately 13 kDa mature GDF-15 (see, for example, Molecular and Cellular Biology, November 2018 Volume 38 Issue 21 e00249-18). In this specification, "GDF-15" may refer to pro-GDF-15 (immature form) or mature GDF-15, and may be used in a sense that includes both.

[0054] The nucleic acid sequence or amino acid sequence of GDF-15 can be identified using known databases (e.g., NCBI, HGNC, ExoCarta, etc.), and is not particularly limited thereto. For example, mammalian GDF-15 can be easily identified by accession numbers such as NM_004864 and NP_004855 for human GDF-15, NM_001330687 and NP_001317616 for mouse GDF-15, and NM_019216 and NP_062089 for rat GDF-15. Furthermore, for example, human GDF-15 can also be identified by IDs such as Entrez Gene_9518, ExoCarta_9518, and UniProt_Q99988.

[0055] "Extracellular vesicles with a high content of GDF-15" refer to extracellular vesicles that express a higher amount of GDF-15 than extracellular vesicles secreted from mesenchymal stem cells under conventional culture conditions (e.g., in the absence of one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharmaceutically acceptable salts thereof), and may be extracellular vesicles that express GDF-15 at 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, or 5 times or more. The amount of GDF-15 expression may be determined appropriately using a known method (e.g., ELISA, Western blotting, chromatography, etc.), and is not particularly limited.

[0056] In one embodiment, the extracellular vesicle population provided by the present invention may be applied to diseases for which mesenchymal stem cells can exert a therapeutic effect, for example, osteochondral diseases such as knee osteoarthritis, liver diseases such as decompensated cirrhosis, autoimmune diseases such as systemic lupus erythematosus, acute graft-versus-host disease and inflammatory bowel diseases such as Crohn's disease, ischemic heart diseases such as myocardial infarction, arteriosclerosis obliterans, critical limb ischemia due to Buerger's disease or collagen disease, and severe lung diseases such as COVID-19, but are not limited to these.

[0057] In one embodiment, the extracellular vesicle population provided by the present invention may be used in the treatment of nervous system diseases. As used herein, the term "nervous system disease" refers to a disease in which certain groups of neurons (e.g., neurons involved in cognitive function or cells involved in motor function) in the brain or spinal cord are damaged, resulting in functional impairment. Examples of nervous system diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Huntington's disease, Alexander disease, cerebrovascular disease, traumatic brain injury, ischemic encephalopathy (e.g., neonatal hypoxic-ischemic encephalopathy (HIE)), central nervous system infection, epilepsy, schizophrenia, and major depression. In one embodiment, the extracellular vesicle population provided by the present invention is preferably applicable to ischemic encephalopathy or traumatic brain injury, more preferably neonatal hypoxic-ischemic encephalopathy (HIE).

[0058] Neonatal hypoxic-ischemic encephalopathy (HIE), one of the major causes of cerebral palsy, is an intractable disease caused by brain tissue damage during fetal or postnatal life. Its primary symptoms are motor and developmental disorders. Despite extensive therapeutic research, no cure has yet been discovered. In recent years, numerous studies have been conducted, primarily using stem cells, on various intractable central nervous system disorders, and numerous therapeutic effects have been reported. However, a clear therapeutic effect on HIE has yet to be demonstrated. A group including the present inventors has previously created HIE model mice and examined the therapeutic effects of umbilical cord blood stem cells (Non-Patent Documents 6-7). However, although a slight tendency toward recovery in motor function was observed, no significant therapeutic effect has yet been demonstrated. The present inventors have demonstrated for the first time that the extracellular vesicle populations provided by the present invention can exert therapeutic effects on traumatic brain injury or ischemic encephalopathy. Therefore, in one embodiment, the extracellular vesicle population provided by the present invention may be preferably applied to traumatic brain injury or ischemic encephalopathy (e.g., neonatal hypoxic-ischemic encephalopathy (HIE)).

[0059] In one embodiment, the present invention provides a method for treating or preventing a nervous system disease in a subject in need thereof, comprising administering to the subject the above-described population of extracellular vesicles.

[0060] All documents mentioned in this specification are incorporated herein by reference in their entirety. The examples of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Modifications of the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, can be made without departing from the spirit of the present invention.

[0061] Example 1: Effect of sulfate compounds on extracellular vesicles (EVs) secreted from umbilical cord-derived mesenchymal stem cells (1)

[0062] Umbilical cord tissue was collected from subjects who provided informed consent, finely chopped, placed in a petri dish, and immersed in a medium (α-MEM) containing 10% bovine serum albumin for 10 to 20 days. The cells that proliferated were then collected (in vitro tissue culture method).

[0063] The collected cells were 5 × 10 ３ / cm ２ The cells were passaged at 100°C, and a portion of the cells at the second passage was confirmed and evaluated as mesenchymal stem cells (MSCs) by flow cytometry using MSC markers. The cells at the third passage were cultured until confluent, and then the medium was replaced with one containing or without sulfatide (22 μM). After further culture for 3 days, the culture supernatant was collected.

[0064] To purify EVs, the culture supernatant was first centrifuged at 2,000 × g for 15 minutes at 4 ° C to remove dead cells and cell debris, and then centrifuged at 12,000 × g for 35 minutes at 4 ° C to remove intracellular organelles from dead cells. Finally, the supernatant was ultracentrifuged at 110,000 × g for 70 minutes at 4 ° C, and the resulting pellet was used as EVs. The prepared EVs were confirmed by electron microscopy and Western blotting using EV marker antibodies (CD9, CD81). The protein content of EVs in the culture supernatant was quantified by the microBCA method, and the particle number of EVs in the culture supernatant was measured using a nanoparticle analysis system, NanoSight (Quantum Design, Japan). The protein content of EVs in the culture supernatant was also quantified by the microBCA method. Furthermore, EVs contained in the culture supernatant were detected by Western blotting using EV marker antibodies (CD9, CD81). β-actin contained in the cell lysate obtained by lysing the MSCs used for EV production was also detected by Western blotting, and the density of the detected bands was quantified using Image-J, normalized by the expression level of β-actin, and compared (Figure 1).

[0065] As a result, it was confirmed that when mesenchymal stem cells were cultured in a medium containing sulfatide, the amount of EVs secreted was promoted (Figure 1).

[0066] Example 2: Effect of sulfate compounds on extracellular vesicles (EVs) secreted from umbilical cord-derived mesenchymal stem cells (2)

[0067] Umbilical cord-derived mesenchymal stem cells were cultured with varying amounts of sulfatide (0 M, 11 μM, or 22 μM) and the effect on the amount of extracellular vesicles (EVs) secreted was observed using the same method as in Example 1. The amount of EVs produced was compared by quantification of protein amount (microBCA method) and Western blot (CD9 antibody).

[0068] As a result, it was revealed that the secretion of EVs from umbilical cord-derived mesenchymal stem cells was promoted in a sulfatide concentration-dependent manner (Figure 2).

[0069] Example 3: Effect of sulfate compounds on extracellular vesicles (EVs) secreted from umbilical cord-derived mesenchymal stem cells (3)

[0070] Umbilical cord-derived mesenchymal stem cells were cultured with various sulfate compounds at 20 μg / mL, and the effect on the amount of extracellular vesicles (EVs) secreted was observed using the same method as in Example 1. The amount of EVs was quantified by protein content (microBCA method) and Western blotting (CD81 antibody).

[0071] As a result, it was revealed that the secretion of EVs from umbilical cord-derived mesenchymal stem cells was promoted not only by sulfatide but also by the addition of cholesterol sulfate (Figure 3).

[0072] Example 4: Effect of sulfate compounds on extracellular vesicles (EVs) secreted from umbilical cord-derived mesenchymal stem cells (4)

[0073] Following the same method as in Example 1, umbilical cord-derived mesenchymal stem cells were cultured for 3 days with or without the addition of sulfatide (22 μM), and extracellular vesicles (EVs) were collected from the resulting culture supernatant by ultracentrifugation or using Mag Capture™ Exosome Isolation Kit PS (Fujifilm Wako Pure Chemical Industries, Ltd., Japan).

[0074] Specifically, ultracentrifugation was performed as follows: The culture supernatant was centrifuged at 2,000 × g for 15 minutes at 4°C to remove dead cells and cell debris, and then further centrifuged at 12,000 × g for 35 minutes at 4°C to remove intracellular organelles from dead cells. This supernatant was then ultracentrifuged at 110,000 × g for 70 minutes at 4°C to obtain a pellet containing EVs.

[0075] EVs were collected using the Mag Capture™ Exosome Isolation Kit PS according to the instructions included with the kit. The Mag Capture™ Exosome Isolation Kit PS is an affinity purification method using Tim4 protein, which specifically recognizes phosphatidylserine (PS) expressed on the surface of extracellular vesicles, particularly exosomes. Therefore, purifying extracellular vesicles using the Mag Capture™ Exosome Isolation Kit PS makes it possible to concentrate extracellular vesicles expressing PS.

[0076] Western blotting was performed on the obtained EVs using antibodies that recognize CD9 and CD81. β-actin contained in the cell lysate obtained by lysing the MSCs used to produce EVs was also detected by Western blotting. The density of the bands detected by Western blotting was quantified using Image-J, normalized by the expression level of β-actin, and compared (Figure 4).

[0077] The CD9 and CD81 contents of extracellular vesicles obtained by various purification methods were examined, and it was found that the recovery of CD9- and CD81-positive extracellular vesicles was reduced when extracellular vesicles obtained with the addition of sulfatide were purified using the Mag Capture™ Exosome Isolation Kit PS (Figure 4). These results suggest that the addition of sulfatide reduces the secretion of PS-positive extracellular vesicles or reduces the expression of CD9 and CD81 on PS-positive extracellular vesicles.

[0078] Example 5: Effect of sulfate compounds on extracellular vesicles (EVs) secreted from umbilical cord-derived mesenchymal stem cells (5)

[0079] Umbilical cord-derived mesenchymal stem cells were cultured with or without the addition of sulfatide (22 μM) according to the same method as in Example 1, and extracellular vesicles (EVs) were recovered from the culture supernatant by ultracentrifugation under the same conditions as in Example 4 or using the Mag Capture™ Exosome Isolation Kit PS (Fujifilm Wako Pure Chemical Industries, Ltd., Japan). An equal amount (20 μl) of purified EVs was loaded onto the plate, and 80 μl of a 20-fold diluted solution of Biotin-Annexin V (Biolegend) was added and allowed to react for 15 minutes at room temperature. After washing twice with Annexin V binding buffer (AVB), 100 μl of a 1000-fold diluted solution of streptavidin-HRP was added and allowed to react for an additional 15 minutes. After washing twice with AVB, the EVs were transferred to a 96-well plate. ELISA reaction solution (3,3',5,5'-tetramethylbenzidine) was added, and the reaction was allowed to proceed at 37°C for 15 minutes. The reaction was stopped with 2N sulfuric acid, and the A450 absorbance was measured. The obtained A450 absorbance was divided by the protein amount (μg / 20 μL) calculated by the microBCA method, and the values ​​(A450 / μg EVs) were compared (Figure 5).

[0080] The PS quantification experiment showed that the addition of sulfatide significantly increased the amount of PS in both EVs collected by ultracentrifugation and those collected using the Mag Capture™ Exosome Isolation Kit. These results, along with those of Example 4, revealed that the addition of sulfatide increased the expression of PS on the surface of EVs and caused a bias in the content of PS, CD9, and CD81 on the surface of EVs (Figure 5).

[0081] Example 6: Comparative verification of the therapeutic effect of extracellular vesicles (EVs) administration on HIE model mice (1)

[0082] Neonatal hypoxic-ischemic encephalopathy (HIE) model mice were created by artificially inducing hypoxia-ischemia by clipping the common carotid artery for one hour in Icr mice (immunodeficient mice) on day 9 of life, which corresponds to the onset of HIE in humans. Successful model creation was confirmed by imaging the brain damage area with MRI and by the decline in motor function in a rotarod test.

[0083] Three weeks after creating a model equivalent to the chronic phase in humans, EVs derived from umbilical cord MSCs were administered via the tail vein. EVs suspended in 100 μl of PBS were administered at 0.1 μg / g body weight. To determine how the administered EVs affect cells surrounding the injury site and contribute to motor function recovery, the location of fluorescently labeled administered EVs in the model mouse brain was tracked, and changes in neurons and glial cells present in the brain were examined by immunostaining brain tissue with various marker antibodies.

[0084] The results showed that sulfatide-treated umbilical cord MSC-derived EVs (Sul(+)-EVs) accumulated at the site of brain injury after tail vein administration (Figures 6 and 7). On the other hand, neither glial scarring nor EV accumulation was observed in mice without brain injury (Figure 8).

[0085] Example 7: Comparative verification of the therapeutic effect of extracellular vesicles (EVs) administration on HIE model mice (2)

[0086] HIE model mice were prepared using the same method as in Example 5, and umbilical cord MSC-derived EVs were administered intranasally. A PBS solution containing umbilical cord MSC-derived EVs obtained with the addition of sulfatide (Sul(+)EVs) at a concentration of 0.108 μg / μL, or a PBS solution containing umbilical cord MSC-derived EVs obtained without the addition of sulfatide (Sul(-)EVs) at a concentration of 0.1 μg / μL, was administered intranasally at a volume of 1 μL per gram of mouse body weight.

[0087] As a result, it was shown that EVs derived from umbilical cord MSCs supplemented with sulfatide (Sul(+)EVs) accumulated at the site of brain injury (glial scar) even after intranasal administration (Fig. 9).On the other hand, EVs derived from umbilical cord MSCs obtained without the addition of sulfatide (Sul(-)EVs) did not accumulate at the site of brain injury (glial scar) in the model mice (Fig. 10).

[0088] Microglia and macrophages are known to recognize and phagocytose PS expressed on the cell surface of apoptotic cells, and it has been reported that large numbers of microglia accumulate in the mouse olfactory bulb (e.g., Non-Patent Documents 4 and 5). Therefore, intranasally administered EVs are likely to be trapped by these cells. However, sulfatide-treated umbilical cord MSC-derived EVs (Sul(+)EVs) secrete EVs with high PS content (PS High EVs) and EVs with low or no PS content (PS Low EVs). When these EVs are intranasally administered to model mice, the PS High EVs are trapped by microglia, which act as a blocker against microglia, allowing the PS Low EVs to reach and accumulate at the site of brain injury (glial scar).

[0089] Based on the results of Examples 1 to 7, it was revealed that sulfatide stimulation not only increased the amount of EVs secreted from umbilical cord MSCs, but also caused a bias in the content of PS, CD9, and CD81 on the surface of EVs. EVs with high PS content were trapped by microglia, but EVs with low PS content escaped this trap and were able to reach the injury site (Figure 11).

[0090] Example 8: Comparative verification of the therapeutic effect of extracellular vesicles (EVs) administration on HIE model mice (3)

[0091] HIE model mice were prepared using the same method as in Example 5, and EVs derived from umbilical cord MSCs cultured with or without sulfatide were administered intranasally. After EV administration, a rotarod test was performed every two weeks to monitor the recovery of motor function. The results showed that HIE model mice administered intranasally with EVs derived from umbilical cord MSCs supplemented with sulfatide (Sul(+)-EVs) recovered motor function to the same level as normal mice after 6 weeks in the rotarod test (Figure 12).

[0092] Example 9: Verification of the neural promoting effect of extracellular vesicles (EVs) produced from umbilical cord-derived mesenchymal stem cells by sulfate compounds on mouse neural stem cells

[0093] Using the same method as in Example 1, umbilical cord-derived mesenchymal stem cells were cultured in medium containing (Sul+) or not containing (Sul-) sulfatide, and a composition (PBS) containing extracellular vesicles (EVs) obtained from the cultured cells was prepared.

[0094] Mouse neural stem cells (NSCs) (1 × 10 ) were plated in a 96-well plate using a medium containing 0.1 μg / well of EVs (Sul+) obtained by culture with sulfatide or EVs (Sul−) obtained by culture without sulfatide. ４ After culturing for 3 days, the number of differentiated neurons was counted by microscopic observation.

[0095] As a result, it was revealed that EVs (sul+) obtained by culture with sulfatide have the effect of significantly promoting neuronal differentiation from mouse neural stem cells (Figure 13).

[0096] Example 10: Search for EVs molecules involved in therapeutic effects (1)

[0097] We compared the components of EVs (Sul+) obtained by culture with sulfatide and EVs (sul-) obtained by culture without sulfatide in an attempt to identify factors directly involved in the treatment.

[0098] RNA was extracted from each EV, and the amount of microRNAs (miR-17, miR-106a, miR-124, miR-133b) reported to be involved in neural regeneration and maturation was quantified using digital PCR (Bio-Rad).

[0099] As a result, it was revealed that miR-17 and miR-106a were contained in higher amounts in EVs (sul+) obtained by culture with sulfatide (Fig. 14).

[0100] Example 11: Effect of the addition of extracellular vesicles (EVs) produced from umbilical cord-derived mesenchymal stem cells on the expression of miR-17 / miR-106a target molecules

[0101] SHSY5Y (neuroblastoma cells) (4 × 10 ５ EVs (sul+) obtained by culture with PBS (-), 4 μg of sulfatide, or EVs (sul-) obtained by culture without sulfatide were added to the culture medium (2 mL / 2000 cells) and cultured for 2 days, and the effect on the expression of PTEN and p38, which are targets of miR-17 and miR-106a, was confirmed by Western blotting.

[0102] As a result, it was revealed that p38 expression was suppressed in SHSY5Y cells cultured for 2 days in a medium containing EVs (sul+) (Fig. 15).

[0103] Example 12: Search for EV molecules involved in therapeutic effects (2) A comparative mass spectrometry analysis was performed on EVs (Sul+) obtained by culture with sulfatide and EVs (Sul-) obtained by culture without sulfatide. It was shown that EVs (Sul+) may contain Growth Differentiation Factor-15 (GDF-15).

[0104] Therefore, Western blotting was performed on EVs (Sul+) obtained by culture with sulfatide or EVs (Sul-) obtained by culture without sulfatide using anti-GDF-15 mouse antibody as the primary antibody and anti-mouse IgG-HRP as the secondary antibody. The density of the detected bands was analyzed and compared using Image-J. At the same time, the amount of protein contained in a 10 μL sample containing EVs was also quantified, and the expression level of GDF-15 per μg of protein was compared.

[0105] As a result, it was revealed that GDF-15 (immature) was significantly increased in EVs obtained with Sul(+) compared to EVs obtained with Sul(-).

[0106] GDF-15 is involved in neural differentiation and neural stem cell neogenesis and has been reported to have therapeutic effects against Alzheimer's disease (e.g., Dong Hyun Kim et al. STEM CELLS AND DEVELOPMENT Volume 24, Number 20, 2015; Yuelin Zhang et al. Scientific Reports, 5:11235, 2015; Wen-Ping Xiong et al. Brain Research Bulletin 177 (2021) 92-102). Therefore, since EVs obtained by Sul(+) had significantly increased GDF-15 (immature), it is possible that the increase in GDF-15 brought about a therapeutic effect on HIE model mice.

[0107] Example 13: Relationship between the amount of sulfatide added to the culture medium and the amount of GDF-15 contained in extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells

[0108] Following the same method as in Example 1, umbilical cord-derived mesenchymal stem cells were cultured with varying amounts of sulfatide added (0 M (PBS), 5.5 μM, 8.25 μM, 11 μM, 16.5 μM, or 22 μM), and the effect on the amount of extracellular vesicles (EVs) secreted was observed. The expression levels of each protein were compared by quantification of protein amount (microBCA method) and Western blotting (anti-GDF-15 antibody, anti-CD81 antibody, CD9 antibody, anti-β-actin antibody).

[0109] As a result, it was revealed that the expression level of GDF-15 in EVs of umbilical cord-derived mesenchymal stem cells increased depending on the sulfatide concentration (FIG. 17).

[0110] Example 14: Relationship between the culture time after addition of sulfatide to the medium and the amount of GDF-15 contained in extracellular vesicles (EVs) obtained from human umbilical cord-derived mesenchymal stem cells

[0111] Following the same method as in Example 1, umbilical cord-derived mesenchymal stem cells were cultured with or without the addition of sulfatide (0 M (PBS) or 16.5 μM), and the amount of GDF-15 in extracellular vesicles (EVs) collected over time was observed. The expression levels of each protein were compared by quantification based on protein amount (microBCA method) and Western blotting (anti-GDF-15 antibody, anti-CD81 antibody, CD9 antibody, anti-β-actin antibody).

[0112] As a result, it was revealed that the expression level of GDF-15 in EVs of umbilical cord-derived mesenchymal stem cells increased depending on the time elapsed after the addition of sulfatide (FIG. 18).

[0113] Example 15: Effect of added sulfate compounds on the amount of GDF-15 contained in extracellular vesicles (EVs) from human umbilical cord-derived mesenchymal stem cells

[0114] Umbilical cord-derived mesenchymal stem cells were cultured in an EVs-free medium containing various sulfate compounds (15 μg / mL) in the same manner as in Example 1, and the amount of GDF-15 in extracellular vesicles (EVs) collected 72 hours later was observed. The expression levels of each protein were compared by quantification based on protein amount (microBCA method) and Western blotting (anti-GDF-15 antibody, anti-CD81 antibody, CD9 antibody, anti-β-actin antibody).

[0115] As a result, the addition of sulfate compounds other than sulfatide did not change the expression level of GDF-15 in EVs of umbilical cord-derived mesenchymal stem cells (FIG. 19).

[0116] Example 16: Effect of added sulfate compounds on the amount of GDF-15 contained in extracellular vesicles (EVs) from human umbilical cord-derived mesenchymal stem cells

[0117] Umbilical cord-derived mesenchymal stem cells were cultured in an EVs-free medium containing various sulfate compounds (15 μg / mL) in the same manner as in Example 1, and the amount of GDF-15 in extracellular vesicles (EVs) collected 72 hours later was observed. The expression levels of each protein were compared by quantification of protein amount (microBCA method) and Western blotting (anti-GDF-15 antibody, anti-CD81 antibody, CD9 antibody, anti-CD63 antibody, and anti-β-actin antibody).

[0118] As a result, the addition of seminolipid, like sulfatide, increased the expression of GDF-15 in EVs from umbilical cord-derived mesenchymal stem cells (Figure 20). However, sphingosine-1-phosphate (S1P), which is generally known to promote the secretion of EVs, did not increase the amount of GDF-15 in EVs (Figure 20).

[0119] Example 17: Effect of sulfatide addition on the amount of GDF-15 contained in extracellular vesicles (EVs) from cells highly expressing GDF-15

[0120] Cells highly expressing GDF-15 were examined in "THE HUMAN PROTEIN ATLAS" (URL: https: / / www.proteinatlas.org / ) (FIG. 21).

[0121] Among the cells identified by the database, experiments were conducted using bone marrow cells, particularly K562 cells, a cell line derived from a leukemia patient. Following the same method as in Example 1, K562 cells were cultured with or without the addition of sulfatide (0 M (PBS) or 16.5 μM), and the expression level of GDF-15 in extracellular vesicles (EVs) was observed. The expression levels of each protein were compared by quantification of protein amount (microBCA method) and Western blotting (anti-GDF-15 antibody, anti-TGF-β antibody, anti-CD81 antibody, CD9 antibody, and anti-β-actin antibody).

[0122] As a result, it was revealed that the expression level of GDF-15 in EVs of K562 cells was increased by the addition of sulfatide (FIG. 22).

[0123] Example 18: Effect of sulfatide addition on the amount of GDF-15 contained in extracellular vesicles (EVs) from cells highly expressing GDF-15

[0124] Following the same method as in Example 1, canine kidney-derived cell lines (MDCK cells, human renal carcinoma-derived cell lines (KMRC-20), L1CAM knockout-KMRC-20 cells, human umbilical cord-derived mesenchymal stem cells, and human neuroblastoma cell lines (SHSY-5Y) were cultured with or without the addition of sulfatide (0 M (PBS) or 16.5 μM), and the expression level of GDF-15 in extracellular vesicles (EVs) was observed. The expression level of each protein was compared by quantification based on protein amount (microBCA method) and Western blotting (anti-GDF-15 antibody). Note that L1CAM knockout-KMRC-20 cells are a cell line in which L1CAM, a molecule that has been proposed as a candidate receptor for sulfatide, has been knocked out.

[0125] As a result, it was revealed that the addition of sulfatide increased the expression level of GDF-15 in EVs, even when using the human renal carcinoma-derived cell line KMRC-20 and the human neuroblastoma cell line SHSY-5Y (Figure 23).Furthermore, the addition of sulfatide increased the expression level of GDF-15 in EVs, even when using a cell line in which L1CAM, a molecule that has been proposed as a candidate receptor for sulfatide, was knocked out (Figure 23).

[0126] Example 19: Effect of sulfatide addition on the amount of GDF-15 contained in extracellular vesicles (EVs) from cells highly expressing GDF-15

[0127] The experiment was basically carried out in accordance with Example 1. Each cell type (human umbilical cord-derived mesenchymal stem cells, K562 cells, KMRC-20 cells, or SHSY-5Y cells) was cultured until 100% confluent. The medium was replaced with one containing sulfatide (16.5 μM), and EVs were collected from the culture supernatant three days later. The amount of protein was quantified using the microBCA method, and 0.2 μg was subjected to Western blotting (anti-GDF-15 antibody).

[0128] As a result, it was found that EVs derived from human umbilical cord-derived mesenchymal stem cells contained the highest amount of GDF-15 (Figure 24).

Claims

1. A method for producing a population of extracellular vesicles including extracellular vesicles with low expression of phosphatidylserine on the membrane surface (low PS extracellular vesicles), comprising: (1) culturing animal cells in a medium containing one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharma- ceutically acceptable salts thereof; and (2) recovering the extracellular vesicle population contained in the medium after carrying out the step (1); A method comprising:

2. The method described in claim 1, wherein the sulfated glycolipids are one or more selected from the group consisting of sulfatides and seminolipids.

3. The method according to claim 1 or 2, wherein the animal cell is a cell that highly expresses Growth Differentiation Factor-15 (GDF-15).

4. The method described in claim 3, wherein the GDF-15 highly expressing cells are mesenchymal cells, female reproductive lineage cells, bone marrow-derived cells, endothelial cells, hepatic cells, renal cancer-derived cells, or neuroblastoma-derived cells.

5. The method according to claim 3, wherein the GDF-15 highly expressing cells are mesenchymal stem cells.

6. The method according to claim 5, wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord, dental pulp, synovium, placenta, and / or fetal membrane, and / or mesenchymal stem cells obtained by inducing differentiation from pluripotent stem cells.

7. The method described in claim 5, wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.

8. A method according to any one of claims 1 to 2, wherein the phosphatidylserine content in the low PS extracellular vesicles is 0.5-fold or less compared to the extracellular vesicles secreted from the animal cells cultured in the absence of the sulfate compound.

9. The method according to any one of claims 1 to 2, wherein the extracellular vesicle population comprises extracellular vesicles in which phosphatidylserine is highly expressed on the membrane surface (high PS extracellular vesicles).

10. The method according to any one of claims 1 to 2, wherein the population of extracellular vesicles is used for the treatment of a nervous system disease.

11. The method according to claim 10, wherein the nervous system disease is ischemic encephalopathy or traumatic brain injury.

12. The method according to any one of claims 1 to 2, wherein the extracellular vesicle population comprises extracellular vesicles having a high content of microRNA selected from the group consisting of miR-17 and miR-106.

13. The method according to any one of claims 1 to 2, wherein the extracellular vesicle population comprises extracellular vesicles having a high content of GDF-15.

14. A population of extracellular vesicles comprising extracellular vesicles with low expression of phosphatidylserine on the membrane surface (low PS extracellular vesicles), obtained by the method according to any one of claims 1 to 2.

15. An extracellular vesicle population comprising extracellular vesicles derived from animal cells that have low expression of phosphatidylserine on the membrane surface (low PS extracellular vesicles).

16. The extracellular vesicle population described in claim 15, wherein the extracellular vesicles are derived from cells that highly express Growth Differentiation Factor-15 (GDF-15).

17. The extracellular vesicle population described in claim 16, wherein the GDF-15 highly expressing cells are mesenchymal stem cells, female germline cells, bone marrow-derived cells, renal cancer-derived cells, or neuroblastoma-derived cells.

18. The extracellular vesicle population described in claim 16, wherein the GDF-15 highly expressing cells are mesenchymal stem cells.

19. The extracellular vesicle population described in claim 18, wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord, dental pulp, synovium, placenta, and / or fetal membrane, and / or mesenchymal stem cells obtained by inducing differentiation from pluripotent stem cells.

20. The extracellular vesicle population of claim 18, wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.

21. The extracellular vesicle population described in any one of claims 15 to 16, wherein the phosphatidylserine content in the low PS extracellular vesicles is 0.5-fold or less compared to the extracellular vesicles secreted from the animal cells cultured in the absence of one or more sulfate compounds selected from the group consisting of sulfated glycolipids, cholesterol sulfate, and pharma- ceutically acceptable salts thereof.

22. The extracellular vesicle population described in any one of claims 15 to 16, wherein the extracellular vesicle population includes extracellular vesicles having a high expression of phosphatidylserine on the membrane surface (high PS extracellular vesicles).

23. The extracellular vesicle population described in any one of claims 15 to 16, wherein the extracellular vesicle population is used for the treatment of a nervous system disease.

24. The extracellular vesicle population of claim 23, wherein the nervous system disease is ischemic encephalopathy or traumatic brain injury.

25. The extracellular vesicle population described in any one of claims 15 to 16, wherein the extracellular vesicle population comprises extracellular vesicles having a high content of microRNA selected from the group consisting of miR-17 and miR-106.

26. The extracellular vesicle population described in any one of claims 15 to 16, wherein the extracellular vesicle population comprises extracellular vesicles having a high content of GDF-15.

27. A method for treating or preventing a nervous system disorder in a subject in need thereof, comprising: Administering to the subject a population of extracellular vesicles according to any one of claims 15 to 16; A method comprising: