Pharmaceutical composition and method for producing same

Abstract

Provided are a novel pharmaceutical composition and a method for producing the pharmaceutical composition. Mesenchymal stem cells that overexpress thrombomodulin on the cell surface are used as an active ingredient of the pharmaceutical composition.

Description

Pharmaceutical composition and method for producing the same

[0001] The present invention relates to a pharmaceutical composition and a method for producing the same.

[0002] Mesenchymal stem cells (MSCs) can be isolated not only from tissues such as bone marrow, adipose tissue, synovium, dental pulp, and periodontal ligament, but also from various tissues such as placenta, umbilical cord blood, and umbilical cord, and can be cultured and proliferated in vitro.

[0003] As a means for culturing mesenchymal stem cells, for example, in addition to using a medium (serum medium) containing fetal bovine serum (FBS), a method of using a medium (serum-free medium) with less contamination of proteins derived from heterologous animals can be mentioned. For example, Patent Documents 1 to 4 describe serum-free media used for culturing mesenchymal stem cells.

[0004] Mesenchymal stem cells have pluripotency and can differentiate not only into mesenchymal cells (for example, osteoblasts, adipocytes, and chondrocytes) but also into non-mesenchymal cells (for example, neural progenitor cells and hepatocytes), and are thus expected to be used as a raw material for producing drugs used in regenerative medicine and / or cell therapy.

[0005] In addition, mesenchymal stem cells not only have the ability to differentiate into various cells, but are also known to have functions of regulating inflammation and immunity in the body and suppressing tissue fibrosis through various factors. Utilizing such functions, in recent years, the utilization of mesenchymal stem cells as new therapeutic cell pharmaceuticals for cartilage damage, traumatic brain injury, graft-versus-host disease (GVHD), liver cirrhosis, and biological tissue damage has been studied.

[0006] International Publication No. 2007 / 080919 International Publication No. 2011 / 111787 International Publication No. 2015 / 016357 International Publication No. 2018 / 123968

[0007] While conventional pharmaceutical compositions containing mesenchymal stem cells can exert desired effects (e.g., therapeutic and preventive effects on diseases), they cannot be said to exert their full potential, and there was room for improvement.

[0008] One aspect of the present invention aims to realize a novel pharmaceutical composition and a method for producing the pharmaceutical composition.

[0009] One aspect of the present invention includes the following: a pharmaceutical composition containing mesenchymal stem cells that overexpress thrombomodulin on the surface of the cells as an active ingredient.

[0010] A method for producing a pharmaceutical composition, comprising a cell manufacturing step for producing mesenchymal stem cells that overexpress thrombomodulin on the surface of the cells, as an active ingredient in the pharmaceutical composition.

[0011] According to one aspect of the present invention, a novel pharmaceutical composition and a method for producing the pharmaceutical composition can be realized.

[0012] Figures 101-104 show the test results confirming the characteristics of mesenchymal stem cells according to the embodiment of the present invention. Figures 201-204 show the test results confirming the effects of mesenchymal stem cells according to the embodiment of the present invention on the survival rate of the administered subjects and on the formation of embolisms and thrombi. Figures 301-303 show the test results confirming the effects of mesenchymal stem cells according to the embodiment of the present invention on the expression of α-SMA and colI. Figures 401-402 show the test results confirming the effects of mesenchymal stem cells according to the embodiment of the present invention on the expression of CD3, CD68, and CD163. Figures 501-502 show the test results confirming the effects of a thrombomodulin expression inducer according to the embodiment of the present invention on the expression of TM. Figures 601-602 show the test results confirming the effects of mesenchymal stem cells according to the embodiment of the present invention on the survival rate of the administered subjects and on the formation of embolisms and thrombi. Figures 701-702 show the test results confirming the effects of mesenchymal stem cells according to the embodiment of the present invention on the formation of embolisms and thrombi. Figures 801-802 show the test results confirming the characteristics of mesenchymal stem cells according to the embodiment of the present invention. Figures 901-902 show the test results confirming the characteristics of mesenchymal stem cells according to the embodiment of the present invention. Figures show the test results confirming the characteristics of mesenchymal stem cells according to the embodiment of the present invention. Figures show the test results confirming the characteristics of mesenchymal stem cells according to the embodiment of the present invention. Figures show the test results confirming the characteristics of mesenchymal stem cells according to the embodiment of the present invention.

[0013] Embodiments of the present invention will be described below. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, all academic and patent documents mentioned herein are incorporated herein by reference. In addition, unless otherwise specified herein, "A to B" representing a numerical range means "A or greater, B or less".

[0014] [1. Pharmaceutical Composition] A pharmaceutical composition according to one embodiment of the present invention contains mesenchymal stem cells that overexpress thrombomodulin on the surface of the cells as an active ingredient.

[0015] When mesenchymal stem cells are used as the active ingredient in a pharmaceutical composition, a large number of mesenchymal stem cells are required. One method for obtaining a large number of mesenchymal stem cells is through subculturing of mesenchymal stem cells.

[0016] When mesenchymal stem cells are subcultured, the amount of proteins thought to be involved in the formation of embolisms and thrombi (e.g., tissue factor and plasminogen activator inhibitor 1) increases, and the risk of developing embolisms and thrombi is thought to increase (see, for example, the examples described later). The pharmaceutical composition according to one embodiment of the present invention can reduce the risk of developing embolisms and thrombi by using mesenchymal stem cells with increased thrombomodulin expression.

[0017] When mesenchymal stem cells are subcultured, their properties change, making it difficult to maintain the quality (e.g., efficacy) of the pharmaceutical composition containing them as an active ingredient. A possible cause of the change in the properties of mesenchymal stem cells is a decrease in thrombomodulin expression (see, for example, the examples described later). The pharmaceutical composition according to one embodiment of the present invention can maintain its quality (e.g., efficacy) by using mesenchymal stem cells with increased thrombomodulin expression.

[0018] Unless otherwise specified herein, "thrombomodulin" refers to thrombomodulin expressed on the surface of cells. This "thrombomodulin" may be native thrombomodulin naturally occurring in nature, or it may be artificially produced thrombomodulin (e.g., recombinant thrombomodulin).

[0019] The origin of the above-mentioned mesenchymal stem cells is not limited. These mesenchymal stem cells may be isolated from various tissues (e.g., bone marrow, fat, synovial membrane, dental pulp, periodontal ligament, placenta, umbilical cord blood, or umbilical cord, etc.).

[0020] The above mesenchymal stem cells may be human-derived mesenchymal stem cells (for example, collected human-derived mesenchymal stem cells), or non-human-derived mesenchymal stem cells (for example, mesenchymal stem cells of cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, or rats).

[0021] The active ingredient of the above pharmaceutical composition is mesenchymal stem cells that overexpress thrombomodulin on the cell surface.

[0022] In the above mesenchymal stem cells, by overexpressing thrombomodulin on the cell surface, not only the repair effect of biological tissue damage but also the therapeutic and preventive effects on embolism and thrombosis can be enhanced.

[0023] In the above mesenchymal stem cells, thrombomodulin is overexpressed. In this specification, the "overexpression" is intended to artificially increase the expression level of thrombomodulin.

[0024] At this time, let the expression level of thrombomodulin that has not been artificially increased be α , １ , １ ,

[0025] and the expression level of thrombomodulin that has been artificially increased be β １ When, for example, β １ > α １ , β １ ≧ 1.5 × α １ , β １ ≧ 2.0 × α １ , β １ ≧ 3.0 × α １ , β １ ≧ 4.0 × α １ , β １ ≧ 5.0 × α １ , β [[ID=4​​​​​​​​​​​​​​​​​​​​​The above-mentioned pharmaceutical composition may be used for the treatment or prevention of at least one selected from the group consisting of embolism (e.g., cerebral infarction or disseminated intravascular coagulation), thrombosis (e.g., cerebral infarction or disseminated intravascular coagulation), inflammation (e.g., acute kidney injury, chronic kidney disease, sepsis, ARDS, alcoholic hepatitis, or non-alcoholic steatohepatitis), fibrosis (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis, or cirrhosis), organ damage (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis, or cirrhosis), tissue damage (e.g., multiple organ failure), immunosuppression (e.g., undesirable immunosuppression (e.g., acute kidney injury, chronic kidney disease, sepsis, ARDS, alcoholic hepatitis, or non-alcoholic steatohepatitis)), and increased apoptosis (e.g., undesirable increased apoptosis (e.g., multiple organ failure)). These symptoms may occur individually or in any combination.

[0026] The above-mentioned pharmaceutical compositions may be used for the treatment or prevention of cerebral infarction, acute kidney injury, chronic kidney disease, sepsis, disseminated intravascular coagulation syndrome, pulmonary fibrosis, idiopathic pulmonary fibrosis, ARDS, cirrhosis, alcoholic hepatitis, non-alcoholic steatohepatitis, or multiple organ failure.

[0027] The diseases described above are those that tend to involve tissue damage, embolism, and / or thrombosis. The pharmaceutical composition according to one embodiment of the present invention has not only a restorative effect on tissue damage, but also therapeutic and preventive effects against embolism and thrombosis, and can therefore effectively treat the diseases described above.

[0028] The administration route of the above-mentioned pharmaceutical composition is not limited and may include, for example, intravascular administration (e.g., intra-arterial administration, intravenous administration) or local administration to the affected area. When the administration route is intravascular administration (e.g., intra-arterial administration, intravenous administration), the pharmaceutical composition can be administered to the body more easily, the therapeutic effect can be obtained better in the body to which the pharmaceutical composition is administered, and the risk of embolism and thrombosis in the body to which the pharmaceutical composition is administered can be reduced. As will be shown in the examples described later, when mesenchymal stem cells are administered intravascularly to the body, there is a high risk of embolism and thrombosis formation. The pharmaceutical composition according to one embodiment of the present invention has the excellent advantage of reducing the risk of embolism and thrombosis formation.

[0029] The above-mentioned mesenchymal stem cells may be cells in which thrombomodulin expression is not reduced (or substantially not reduced) regardless of the number of cell divisions or passages. The above-mentioned mesenchymal stem cells have the excellent advantage of being able to better reduce the risk of embolism and thrombosis, and to better maintain the quality of the pharmaceutical composition (e.g., efficacy), even with an increased number of cell divisions and / or passages.

[0030] For example, consider mesenchymal stem cells that have undergone a desired number of cell divisions (e.g., 1 or more, 3 or more, 5 or more, 7 or more). The expression level of thrombomodulin in mesenchymal stem cells before the desired number of cell divisions is A. １ The expression level of thrombomodulin in mesenchymal stem cells after the desired number of cell divisions is B １ Let's assume that, for example, 0.6 × A １ ≤ B １ , 0.7 × A １ ≤ B １ , 0.8 × A １ ≤ B １ , 0.9 × A １ ≤ B １ , or 1.0 × A １ ≤ B １ This is also possible. Furthermore, the upper limit of the number of cell divisions is not limited and could be, for example, 100, 50, 40, 30, 20, or 10.

[0031] For example, consider mesenchymal stem cells that have undergone a desired number of passages (e.g., 1 or more, 3 or more, 5 or more, 7 or more). The expression level of thrombomodulin in the mesenchymal stem cells before the desired number of passages is A. ２ The expression level of thrombomodulin in mesenchymal stem cells after the desired number of passages is B ２ Let's assume that, for example, 0.6 × A ２ ≤ B ２ , 0.7 × A ２ ≤ B ２ , 0.8 × A ２ ≤ B ２ , 0.9 × A ２ ≤ B ２ , or 1.0 × A ２ ≤ B ２ This is also acceptable. Furthermore, there is no upper limit to the number of successive generations; for example, it could be 100, 50, 40, 30, 20, or 10 times.

[0032] The above-mentioned mesenchymal stem cells are preferably mesenchymal stem cells into which a thrombomodulin expression vector has been introduced, or mesenchymal stem cells that have been in contact with a thrombomodulin expression inducer. With this configuration, mesenchymal stem cells that overexpress thrombomodulin on the cell surface can be easily realized.

[0033] The vector for thrombomodulin expression is not limited to any vector that can be introduced into mesenchymal stem cells and may be a vector into which a polynucleotide encoding thrombomodulin can be inserted downstream of a desired promoter so that thrombomodulin can be expressed within the mesenchymal stem cells.

[0034] As an example of the above polynucleotide, any of the following polynucleotides (1) to (3) can be cited. However, the present invention is not limited to these polynucleotides: (1) a polynucleotide consisting of the base sequence shown in "accession number: NC_000020"; (2) a polynucleotide that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the polynucleotide consisting of the base sequence shown in "accession number: NC_000020" and encodes a polypeptide having anticoagulant activity and / or anti-inflammatory activity in blood; or (3) a polynucleotide that has 90% or more (or 95% or more, 98% or more) sequence identity with the polynucleotide consisting of the base sequence shown in "accession number: NC_000020" and encodes a polypeptide having anticoagulant activity and / or anti-inflammatory activity in blood.

[0035] In this specification, "stringent conditions" refer to conditions under which so-called sequence-specific double-stranded polynucleotides are formed, and non-specific double-stranded polynucleotides are not formed. In other words, it can also be described as conditions under which highly homologous nucleic acids, for example, hybridize at a temperature range of 15°C, preferably 10°C, and even more preferably 5°C lower than the melting temperature (Tm value) of a perfectly matched hybrid.

[0036] For example, one example is 0.25 M Na ２ HPO ４ Hybridize for 16 to 24 hours in a buffer solution consisting of pH 7.2, 7% SDS, 1 mM EDTA, and 1 × Denhardt's solution at a temperature of 60 to 68°C, preferably 65°C, and more preferably 68°C, and then add 20 mM Na ２ HPO ４ One possible condition is to perform two 15-minute washes in a buffer solution consisting of pH 7.2, 1% SDS, and 1 mM EDTA at a temperature of 60 to 68°C, preferably 65°C, and more preferably 68°C.

[0037] Another example involves performing pre-hybridization overnight at 42°C in a hybridization solution containing 25% formamide, or more severely, 50% formamide, 4×SSC (sodium chloride / sodium citrate), 50 mM Hepes pH 7.0, 10× Denhardt solution, and 20 μg / mL denatured salmon sperm DNA. After this, the labeled probe is added, and hybridization is performed by incubating at 42°C overnight. Subsequent washing conditions can be approximately "1×SSC, 0.1% SDS, 37°C", more severely "0.5×SSC, 0.1% SDS, 42°C", and even more severely "0.2×SSC, 0.1% SDS, 65°C". Thus, the more stringent the washing conditions during hybridization, the more specific the hybridization becomes. However, the combinations of SSC, SDS, and temperature conditions described above are illustrative, and those skilled in the art can achieve similar stringency by appropriately combining the aforementioned or other elements that determine the stringency of hybridization (e.g., probe concentration, probe length, hybridization reaction time, etc.). This is described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001).

[0038] The identity of nucleotide sequences and amino acid sequences can be determined using programs such as BLASTX (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). This program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). When analyzing amino acid sequences using BLASTX, the parameters should be, for example, score = 50 and wordlength = 3. Alternatively, when analyzing amino acid sequences using the Gapped BLAST program, the procedure can be performed as described by Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). The specific methods for these analyses are publicly known. Additions or deletions (e.g., gaps) may be permitted to align the comparison base sequence or amino acid sequence to an optimal state.

[0039] Whether or not a polypeptide is a "polypeptide having anticoagulant activity and / or anti-inflammatory activity in blood" can be confirmed by known methods. For example, after expressing the desired polypeptide in the desired cells, the increase or decrease in the expression of genes that serve as indicators of anticoagulant activity and / or anti-inflammatory activity can be confirmed in those cells.

[0040] The expression inducer described above can be any substance that induces thrombomodulin expression in mesenchymal stem cells when brought into contact with them, and its specific composition is not limited.

[0041] The expression inducer is preferably BMS-345541 (Merck 401480, 4-(2'-Aminoethyl)amino-1,8-dimethylimidazo[1,2-a]quinoxaline), an IKKβ inhibitor, a compound having an imidazopyrazine skeleton in its molecule, an IκB inducer, or a compound having a flavonoid skeleton in its molecule.

[0042] The above IKKβ inhibitors are not limited to those mentioned above, but include, for example, TPCA-1, IKK-16, LY2409881, and BAY-985.

[0043] The compounds having an imidazopyrazine skeleton within the above molecule are not limited to those mentioned above, and examples include N-butan-2-yl-1-methyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-amine (PubChem CID; 3583535), N-(3-methylbutyl)-[1,2,4]triazolo[4,3-a]quinoxalin-4-amine (PubChem CID; 6622885), 1-(1,8-Dimethylimidazo[1,2-a]quinoxalin-4-yl)ethane-1,2-diamine (PubChem CID; 124159894), and 4-(2-Aminoethyl)-1,8-dimethylimidazo[1,2-a]quinoxalin-2-amine (PubChem CID; 140554219).

[0044] The above-mentioned IκB inducers are not limited to those mentioned above, and examples include WS3 (CAS No. 1421227-52-2) and WS6 (CAS No. 1421227-53-3). These IκB inducers include those that promote IκB expression and / or function.

[0045] The compounds having a flavonoid skeleton within the above molecule are not limited to, but include, for example, flavones (e.g., baicalin), flavanols (e.g., epigallocatechin allate (EGCG)), and flavonols (e.g., quercetin).

[0046] The above-mentioned mesenchymal stem cells are preferably mesenchymal stem cells cultured in serum-free medium. With this configuration, mesenchymal stem cells that overexpress thrombomodulin on the cell surface can be easily realized.

[0047] The composition of the serum-free medium described above is not limited, and known serum-free media can be used as appropriate. Examples of known serum-free media include STK1 (TwoCell Co., Ltd.), STK2 (TwoCell Co., Ltd.), Human Mesenchymal Stem Cell Dedicated Complete Synthetic Medium Kit (MSCGM-CD Bullet Kit) (Lonza), Mesenchymal Stem Cell Growth Medium DXF (Ready-touse) (PromoCell GmbH.), Stem Pro MSC SFM Xeno free (Thermo Fisher Scientific Inc.), and MesenCult-ACF Medium Kit (STEMCELL Technologies Inc.).

[0048] The number of mesenchymal stem cells that overexpress thrombomodulin on their cell surface and are contained in the above pharmaceutical composition is not limited. Mesenchymal stem cells that overexpress thrombomodulin on their cell surface have a low risk of developing embolism and thrombosis. Therefore, the pharmaceutical composition according to one embodiment of the present invention can contain a large amount of mesenchymal stem cells, thereby reducing the risk of developing embolism and thrombosis while increasing the therapeutic and preventive effects of the disease.

[0049] The number of mesenchymal stem cells that overexpress thrombomodulin on the surface of the cells contained in the above pharmaceutical composition is, for example, 5 × 10⁶ per kg of body weight of the recipient. ５ ~7 x 10 ５ cells / kg, 7×10 ５ ~9 x 10 ５ cells / kg, 9×10 ５ ~11 x 10 ５ cells / kg, 11×10 ５ ~13 x 10 ５ cells / kg, or 13 × 10 ５ ~15 x 10 ５ The number of cells / kg may be sufficient to administer mesenchymal stem cells.

[0050] For example, in (i) mesenchymal stem cells into which a thrombomodulin expression vector has been introduced, or mesenchymal stem cells after contact with a thrombomodulin expression inducer, and (ii) mesenchymal stem cells cultured in serum-free medium, it is thought that the expression of a vast number of genes and / or proteins changes to produce the desired effect. In this case, identifying the genes and / or proteins that are important to the present invention from among this vast number of genes and / or proteins would require conducting an unrealistic number of experiments, resulting in excessively large economic expenditures, and it would also be difficult to comprehensively express the results in the claims. From this viewpoint, one embodiment of the present invention includes inventions in which it is generally unrealistic to "directly identify the object by its structure or properties at the time of filing the application."

[0051] [2. Method for Manufacturing Pharmaceutical Compositions] In the following sections, the matters described in [1. Pharmaceutical Compositions] above will generally be omitted from further explanation.

[0052] A method for producing a pharmaceutical composition according to one embodiment of the present invention comprises a cell manufacturing step of producing mesenchymal stem cells that overexpress thrombomodulin on the surface of the cells, as the active ingredient of the pharmaceutical composition.

[0053] The above-mentioned pharmaceutical composition may be used for the treatment or prevention of at least one selected from the group consisting of embolism (e.g., cerebral infarction or disseminated intravascular coagulation), thrombosis (e.g., cerebral infarction or disseminated intravascular coagulation), inflammation (e.g., acute kidney injury, chronic kidney disease, sepsis, ARDS, alcoholic hepatitis, or non-alcoholic steatohepatitis), fibrosis (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis, or cirrhosis), organ damage (e.g., pulmonary fibrosis, idiopathic pulmonary fibrosis, or cirrhosis), tissue damage (e.g., multiple organ failure), immunosuppression (e.g., undesirable immunosuppression (e.g., acute kidney injury, chronic kidney disease, sepsis, ARDS, alcoholic hepatitis, or non-alcoholic steatohepatitis)), and increased apoptosis (e.g., undesirable increased apoptosis (e.g., multiple organ failure)). These symptoms may occur individually or in any combination.

[0054] The above-mentioned pharmaceutical compositions may be used for the treatment or prevention of cerebral infarction, acute kidney injury, chronic kidney disease, sepsis, disseminated intravascular coagulation syndrome, pulmonary fibrosis, idiopathic pulmonary fibrosis, ARDS, cirrhosis, alcoholic hepatitis, non-alcoholic steatohepatitis, or multiple organ failure.

[0055] The route of administration of the above pharmaceutical composition is not limited and may include, for example, intravascular administration (e.g., intra-arterial administration, intravenous administration) or local administration to the affected area.

[0056] The above-mentioned mesenchymal stem cells may be cells in which thrombomodulin expression is not reduced (or substantially not reduced) regardless of the number of cell divisions or passages.

[0057] The above cell manufacturing process preferably includes introducing a thrombomodulin expression vector into mesenchymal stem cells, or contacting mesenchymal stem cells with a thrombomodulin expression inducer. With this configuration, mesenchymal stem cells that overexpress thrombomodulin on the cell surface can be easily realized.

[0058] Furthermore, the method for introducing the expression vector into mesenchymal stem cells is not limited; any known introduction method appropriate to the type of expression vector can be used. The method for contacting the mesenchymal stem cells with the expression inducer is also not limited; for example, the expression inducer can be added to the culture medium in which the mesenchymal stem cells are cultured.

[0059] The expression inducer is preferably BMS-345541, an IKKβ inhibitor, a compound having an imidazopyrazine skeleton in its molecule, an IκB inducer, or a compound having a flavonoid skeleton in its molecule.

[0060] The above cell manufacturing process preferably includes culturing mesenchymal stem cells in serum-free medium.

[0061] Details regarding the serum-free medium were explained in section [1. Pharmaceutical Composition] above, so that explanation will be omitted here.

[0062] [3. Others] <1> A method for treating or preventing a disease, comprising an administration step of administering a pharmaceutical composition containing mesenchymal stem cells that overexpress thrombomodulin on the surface of cells as an active ingredient to a subject (e.g., a human or a non-human animal).

[0063] <2> The treatment or prevention method described in <1>, wherein the above disease is a disease characterized by at least one selected from the group consisting of embolism, thrombosis, inflammation, fibrosis, organ damage, tissue damage, increased immunity, and increased apoptosis.

[0064] <3> The above-mentioned diseases are cerebral infarction, acute kidney injury, chronic kidney disease, sepsis, disseminated intravascular coagulation syndrome, pulmonary fibrosis, idiopathic pulmonary fibrosis, ARDS, cirrhosis, alcoholic hepatitis, non-alcoholic steatohepatitis, or multiple organ failure, and the treatment or prevention method described in <2>.

[0065] <4> In the above administration step, the above pharmaceutical composition is administered intravascularly, the therapeutic or preventive method according to any one of <1> to <3>.

[0066] <5> The above-mentioned mesenchymal stem cells are cells in which thrombomodulin expression is not reduced regardless of the number of cell divisions or passages, the treatment or prevention method described in any of <1> to <4>.

[0067] <6> The therapeutic or preventive method according to any one of <1> to <5>, wherein the mesenchymal stem cells are mesenchymal stem cells into which a thrombomodulin expression vector has been introduced, or mesenchymal stem cells after contact with a thrombomodulin expression inducer.

[0068] <7> The treatment or prevention method according to <6>, wherein the expression inducer is BMS-345541, an IKKβ inhibitor, a compound having an imidazopyrazine skeleton in its molecule, an IκB inducer, or a compound having a flavonoid skeleton in its molecule.

[0069] <8> The above-mentioned mesenchymal stem cells are mesenchymal stem cells cultured in serum-free medium, the treatment or prevention method described in any of <1> to <7>.

[0070] This invention may also contribute to achieving Goal 3 of the United Nations' Sustainable Development Goals (SDGs), "Ensure healthy lives and promote well-being for all."

[0071] [4. Summary] One aspect of the present invention includes the following aspects: [1] A pharmaceutical composition containing mesenchymal stem cells that overexpress thrombomodulin on the surface of the cells as an active ingredient.

[0072] [2] The pharmaceutical composition according to [1], wherein the pharmaceutical composition is used for the treatment or prevention of at least one selected from the group consisting of embolism, thrombosis, inflammation, fibrosis, organ damage, tissue damage, immunosuppression, and apoptosis enhancement.

[0073] [3] The pharmaceutical composition described in [2], which is used for the treatment or prevention of cerebral infarction, acute kidney injury, chronic kidney disease, sepsis, disseminated intravascular coagulation syndrome, pulmonary fibrosis, idiopathic pulmonary fibrosis, ARDS, cirrhosis of the liver, alcoholic hepatitis, non-alcoholic steatohepatitis, or multiple organ failure.

[0074] [4] The pharmaceutical composition described in any of [1] to [3] above, which is administered intravascularly.

[0075] [5] The mesenchymal stem cells are cells in which thrombomodulin expression is not reduced regardless of the number of cell divisions or passages, according to any one of [1] to [4].

[0076] [6] The pharmaceutical composition according to any one of [1] to [5], wherein the mesenchymal stem cells are mesenchymal stem cells into which a thrombomodulin expression vector has been introduced, or mesenchymal stem cells after contact with a thrombomodulin expression inducer.

[0077] [7] The pharmaceutical composition according to [6], wherein the expression inducer is BMS-345541, an IKKβ inhibitor, a compound having an imidazopyrazine skeleton in its molecule, an IκB inducer, or a compound having a flavonoid skeleton in its molecule.

[0078] [8] The pharmaceutical composition according to any one of [1] to [7], wherein the mesenchymal stem cells are mesenchymal stem cells cultured in serum-free medium.

[0079] [9] A method for producing a pharmaceutical composition, comprising a cell manufacturing step for producing mesenchymal stem cells that overexpress thrombomodulin on the surface of cells as an active ingredient of the pharmaceutical composition.

[0080]

[10] A method for producing the pharmaceutical composition according to [9], wherein the pharmaceutical composition is used for the treatment or prevention of at least one selected from the group consisting of embolism, thrombosis, inflammation, fibrosis, organ damage, tissue damage, immunosuppression, and apoptosis enhancement.

[0081]

[11] A method for producing the pharmaceutical composition described in

[10] , wherein the pharmaceutical composition is used for the treatment or prevention of cerebral infarction, acute kidney injury, chronic kidney disease, sepsis, disseminated intravascular coagulation syndrome, pulmonary fibrosis, idiopathic pulmonary fibrosis, ARDS, cirrhosis of the liver, alcoholic hepatitis, non-alcoholic steatohepatitis, or multiple organ failure.

[0082]

[12] A method for producing the pharmaceutical composition described in any of [9] to

[11] , wherein the pharmaceutical composition is administered intravascularly.

[0083]

[13] The above-mentioned mesenchymal stem cells are cells in which thrombomodulin expression is not reduced regardless of the number of cell divisions or passages, a method for producing the pharmaceutical composition according to any one of [9] to

[12] .

[0084]

[14] A method for producing a pharmaceutical composition according to any one of [9] to

[13] , wherein the cell production step includes introducing a vector for thrombomodulin expression into mesenchymal stem cells, or contacting mesenchymal stem cells with a thrombomodulin expression inducer.

[0085]

[15] The method for producing the pharmaceutical composition according to

[14] , wherein the expression inducer is BMS-345541, an IKKβ inhibitor, a compound having an imidazopyrazine skeleton in its molecule, an IκB inducer, or a compound having a flavonoid skeleton in its molecule.

[0086]

[16] A method for producing a pharmaceutical composition according to any one of [9] to

[15] , wherein the cell production step includes culturing mesenchymal stem cells in serum-free medium.

[0087] <1. Overview of the Test Method> <1-1. Animals> Male Sprague-Dawley rats (SD rats) (6-7 weeks old) were purchased from Charles River Laboratories Japan (Yokohama, Japan). When the rats were 8 weeks old, an ischemic reperfusion injury (IRI) model animal was created using these rats. The body weight of the rats used in the following tests was approximately 300 g.

[0088] <1-2. Isolation and Culture of Adipose Mesenchymal Stem Cells (ASCs)> ASCs were isolated from the adipose tissue of patients (44-57 years old) who underwent breast reconstruction surgery. These ASCs were cultured in STK2 medium (37415-08, Kanto Chemical, Tokyo, Japan), a serum-free medium, and used in the test.

[0089] <1-3. Production of Thrombomodulin Overexpressing ASCs Using Adeno-Associated Virus (AAV)> Thrombomodulin (TM) cDNA (accession number: NC_000020) and Enhanced Green Fluorescent Protein (EGFP) cDNA (accession number: MN832871) were synthesized by outsourcing (eurofins genomics, Tokyo, Japan). These cDNAs were subcloned into the multi-cloning site of the pAAV-CMV vector using restriction enzymes (EcoRI and SalI) (Takara Bio Inc., Shiga, Japan).

[0090] AAVpro® 293T Cells (Takara) were cultured at 37°C in a 10 cm diameter dish using DMEM (Sigma Aldrich, no antibiotics added) containing 10% FBS.

[0091] After subculturing the above AAVpro® 293T cells, they were cultured until they reached approximately 70% confluence. Subsequently, the pAAV-CMV vector, pRC6 vector, and pHelper vector, which are used to express the target gene, were introduced into the above AAVpro® 293T cells using TrasIT-293 Transfection Reagent (Takara) according to the attached protocol.

[0092] Twenty-four hours after introducing the above vector, the culture medium was changed from DMEM containing 10% FBS (Sigma Aldrich, no antibiotics added) to DMEM containing 2% FBS (Sigma Aldrich, no antibiotics added).

[0093] After culturing the above AAVpro® 293T cells for a further 24 hours (48 hours after vector introduction), the AAV solution was purified and concentrated using the AAVpro® Purification Kit Maxi All Serotypes (Takara). The virus concentration in the purified and concentrated AAV solution was measured using the AAVpro® Titration Kit (for Real Time PCR) Ver. 2 (Takara). The purified and concentrated AAV solution was stored at -80°C.

[0094] When ASCs cultured on a 10 cm diameter dish reached a 60-70% confluence, an AAV solution was added to the culture medium. ASCs expressing TM and EGFP were designated ASC-TM and ASC-EGFP, respectively. On the other hand, ASCs infected with AAV that did not express the target gene were designated ASC-null for comparison.

[0095] ASCs were collected 48 hours after the AAV solution was added to the culture medium, and these ASCs were used in the experiment.

[0096] <1-4. TM expression test by pretreatment with IKKβ inhibitor> ASCs cultured in STK2 medium until they reached a confluence of approximately 70% were treated with an IKKβ inhibitor (BMS-345541, Sigma Aldrich) dissolved in DMSO. ASCs stimulated with the IKKβ inhibitor BMS-345541 were designated as ASC-BMS, and ASCs not stimulated with the IKKβ inhibitor BMS-345541 were designated as ASC-NS. ASCs were collected 24 hours after stimulation with the IKKβ inhibitor and used in the test.

[0097] <1-5. Preparation of IRI Model Animals> Rats were anesthetized by intraperitoneal injection of a three-component anesthetic solution consisting of medetomidine, midazolam, and butorfarc. The rats were opened and the left kidney was exposed. The left renal artery was clamped for 60 minutes using a non-traumatic vascular clip. After reperfusion, the left renal artery was clamped at the cranial and caudal abdominal arteries, and ASC (5 × 10) dissolved in 0.2 mL of PBS was injected. ５ Cells (rat) was injected near the left renal artery bifurcation. After a desired number of days had elapsed since injection (e.g., 7 or 21 days), the left kidney of the rats was retrieved and evaluated for inflammation and fibrosis.

[0098] Furthermore, inflammation is a symptom that can occur in acute kidney injury, chronic kidney disease, sepsis, ARDS, alcoholic hepatitis, and non-alcoholic steatohepatitis. On the other hand, fibrosis is a symptom that can occur in pulmonary fibrosis, idiopathic pulmonary fibrosis, and cirrhosis. In other words, it is thought that by using this IRI model animal, it is possible to evaluate not only inflammation and fibrosis, but also acute kidney injury, chronic kidney disease, sepsis, ARDS, alcoholic hepatitis, non-alcoholic steatohepatitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, and cirrhosis.

[0099] <1-6. Evaluation of rat mortality by transarterial ASC injection> Rats were anesthetized by intraperitoneal injection of a three-component mixture of medetomidine, midazolam, and butorfar. The rats were then laparotomyed and the right kidney was removed.

[0100] One week after removing the right kidney of a rat, the rat was intraperitoneally injected with a three-component anesthetic mixture, the rat was laparotomyed, and the left kidney was exposed. The left renal artery was occluded for 45 minutes using a non-traumatic vascular clip. After reperfusion, with the left renal artery clamped at the cranial and caudal abdominal arteries, ASC-TM or ASC-null (5 × 10) dissolved in 0.2 mL of PBS was administered. ５ Cells (a rat drug) was injected near the left renal artery bifurcation. The number of surviving rats was counted for 21 days after injection.

[0101] <1-7. Evaluation of rat mortality by intravenous ASC injection> ASC-TM, ASC-BMS, or ASC-NS (7.5 × 10⁶ each) dissolved in 1 mL of PBS. ６ Cells (rat) were slowly injected into the tail vein. The number of rat deaths within 24 hours after injection was counted. Most rats died within 30 minutes after injection. Both lungs were collected from dead rats, or from rats that survived 24 hours after injection and were sacrificed at that time. The lungs were shaken in 10% formalin for 24 hours, then fixed with paraffin, and subsequently sectioned into 4 μm thick sections. After removing the paraffin from the sections, they were stained with hematoxylin and eosin (H&E) according to a standard protocol. The number of intravascular thrombi in 10 fields of view per rat was counted.

[0102] <1-8. Differentiation Induction Test of ASCs> ASC-NS, ASC-null, ASC-TM, or ASC-BMS were cultured for 14 days in adipocyte differentiation induction medium (Takara) or myelocyte differentiation induction medium (Sigma-Aldrich) according to the protocol attached to the medium. Oil Red O (Sigma-Aldrich) and Alizarin Red S (FUJIFILM Wako pure Chemical) were used to evaluate adipocyte differentiation and myelocyte differentiation.

[0103] <2. Test Results A> <2-1. Test 1> The ASCs obtained in section <1-2> above were cultured using STK2 medium (37415-08, Kanto Chemical, Tokyo, Japan), which is a serum-free medium.

[0104] ASCs that had been passaged three times (passage 3: P3), five times (passage 5: P5), and seven times (passage 7: P7) were collected, and the collected ASCs were dissolved using cell lysis buffer (Cell Signaling).

[0105] After quantifying the proteins in the obtained lysis solutions, thrombomodulin (TM), tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) contained in each solution were detected by Western blotting.

[0106] For detecting TM, TF, PAI-1, and GAPDH, the following primary antibodies were used: rabbit monoclonal anti-thrombomodulin antibody (abcam, ab109189), rabbit monoclonal anti-tissue factor antibody (abcam, ab228968), rabbit monoclonal anti-PAI-1 antibody (abcam, ab187262), and mouse monoclonal anti-GAPDH antibody (Sigma-Aldrich, G8795), respectively.

[0107] The test results are shown in Figure 1, section 101. As is clear from Figure 1, section 101, the expression levels of TF and PAI-1, which are thought to increase the risk of embolism and thrombosis (e.g., the risk of cerebral infarction and disseminated intravascular coagulation), increased with increasing passage numbers. On the other hand, the expression level of TM decreased with increasing passage numbers.

[0108] <2-2. Experiment 2> The ASCs obtained in section <1-2> above were cultured using STK2 medium (37415-08, Kanto Chemical, Tokyo, Japan), which is a serum-free medium.

[0109] The culture medium in which ASCs are being cultured contains a multiplicity of infection (MOI) of 0.3 × 10⁻⁶ ３ , 1.0 × 10 ３ , 3.0 x 10 ３ , 10 x 10３ , or 30 x 10 ３ To achieve this, the AAV solution for expressing TM obtained in section <1-3> above was added. This infected the ASC with AAV for expressing TM.

[0110] Forty-eight hours after infection with AAV, the percentage of ASCs expressing CD141, the endothelial cell receptor for TM, was confirmed by FACS analysis. Anti-human CD141 IgG antibody (BioLegend, 344103) was used as the primary antibody to detect CD141.

[0111] The test results are shown in Figure 1, section 102. As is clear from Figure 1, section 102, the MOI is 10 × 10 ３ In this case, CD141 expression was confirmed in 99.2% of ASCs. Also, the MOI was 10 × 10 ３ In the above cases, growth impairment and / or cell death were observed in the ASC.

[0112] <2-3. Experiment 3> The ASCs obtained in section <1-2> above were cultured using STK2 medium (37415-08, Kanto Chemical, Tokyo, Japan), which is a serum-free medium.

[0113] The culture medium in which ASCs are being cultured contains a multiplicity of infection (MOI) of 0.3 × 10⁻⁶ ３ , 1.0 × 10 ３ , 3.0 x 10 ３ , 10 x 10 ３ , or 30 x 10 ３ To achieve this, the AAV solution for expressing TM obtained in section <1-3> above was added. This infected the ASC with AAV for expressing TM.

[0114] ASCs were collected 48 hours after infection with AAV, and the mRNA and protein levels of TM in the ASCs were evaluated by RT-PCR and Western blotting.

[0115] Figure 1, section 103 shows the test results for TM mRNA levels, and Figure 1, section 104 shows the test results for TM protein levels. As is clear from Figures 1, sections 103 and 104, the amount of TM mRNA and TM protein levels increased as the MOI increased.

[0116] <3. Test Results B> <3-1. Test 1> The rat mortality rate by transarterial ASC injection was evaluated according to section <1-6> above. It was assumed that if renal infarction occurred in the remaining kidney, the rat would die due to renal failure.

[0117] The test results are shown in Figure 2, item 201. As is clear from Figure 2, item 201, in the group administered ASC-null, 4 out of 11 rats died by day 3, but no rat deaths were observed in the group administered ASC-TM.

[0118] <3-2. Experiment 2> The rat mortality rate induced by intravenous ASC injection was evaluated according to section <1-7> above.

[0119] The test results are shown in Figure 2, section 202. In the group administered ASC-NS, 2 out of 12 rats died within 24 hours, and intravascular pulmonary thrombi were confirmed in 24 out of 120 fields of view by H&E staining. On the other hand, in the group administered ASC-TM, no rats died within 24 hours, and intravascular pulmonary thrombi were confirmed in 3 out of 120 fields of view by H&E staining. The number of intravascular pulmonary thrombi was lower in the group administered ASC-TM compared to the group administered ASC-NS.

[0120] <3-3. Test 3> In accordance with section <1-7> above, administer ASC-EGFP (7.5 × 10) transvenously. ６ After administering cells (for rats) to rats, lung tissue was stained. For staining EGFP and CD41, Living Colors® EGFP Monoclonal Antibody (takara, 632569) and rabbit monoclonal anti-CD41 antibody (abcam, ab181582) were used as primary antibodies, respectively.

[0121] The test results are shown in Figure 2, section 203. As is clear from Figure 2, section 203, immunohistochemical staining confirmed EGFP staining in the area corresponding to the site of pulmonary thrombus formation, and also confirmed staining of CD41, a platelet surface marker. In other words, thrombus formation was confirmed around ASC-EGFP.

[0122] <3-4. Test 4> In accordance with section <1-7> above, administer PBS, ASC-NS (5.0 x 10) intravenously. ５ ASC-TM (5.0 × 10) prepared with cells / rat, MOI = 10000 ５ Rats were administered cells (for rats). Subsequently, rats administered PBS, ASC-NS, and ASC-TM were subjected to H&E staining and CD41 immunostaining.

[0123] The test results are shown in Figure 2, section 204. As is clear from Figure 2, section 204, in rats administered ASC-NS, CD41 staining was observed at the site of thrombus formation. On the other hand, in rats administered ASC-TM, similar to rats administered PBS, neither thrombus nor CD41 staining was observed.

[0124] <4. Test Results C> Following the IRI (ischemic time 60 minutes) of the left kidney as described in section <1-5> above, PBS, ASC-null, or ASC-TM (5.0 x 10) were administered via the abdominal artery. ５ Cells (a rat drug) was injected. Twenty-one days after injection, the left kidney was retrieved, and the expression levels of α-SMA and collagen type I (colI) in the retrieved left kidney were examined by Western blotting, immunohistochemical staining, and Masson's trichrome staining. Note that the expression of α-SMA and extracellular matrix (e.g., collagen type I) serves as an indicator of renal fibrosis.

[0125] For staining α-SMA and colI, mouse monoclonal α-SMA antibody (Sigma-Aldrich, A2547) and rabbit monoclonal anti-collagen I antibody (abcam, ab34710) were used as primary antibodies, respectively.

[0126] The test results are shown in Figures 301, 302, and 303.

[0127] Figure 3, section 301 shows the results of the Western blotting test. As is clear from Figure 3, section 301, the expression level of α-SMA increased significantly in rats administered with PBS. On the other hand, in rats administered with ASC-null, an inhibitory effect on the increase in α-SMA expression was observed compared to rats administered with PBS. In rats administered with ASC-TM, a further inhibitory effect on the increase in α-SMA expression was observed compared to rats administered with ASC-null.

[0128] Figure 3, section 302 shows the results of immunohistochemical staining. As is clear from Figure 3, section 302, the expression levels of α-SMA and COLI increased significantly in rats administered with PBS. On the other hand, in rats administered with ASC-null, an inhibitory effect on the increase in α-SMA and COLI expression was observed compared to rats administered with PBS. In rats administered with ASC-TM, a further inhibitory effect on the increase in α-SMA and COLI expression was observed compared to rats administered with ASC-null.

[0129] Figure 3, section 303 shows the results of the Masson's trichrome staining test. As is clear from Figure 3, section 303, the expression levels of α-SMA and colI increased significantly in rats administered with PBS. On the other hand, in rats administered with ASC-null, an inhibitory effect on the increase in α-SMA and colI expression was observed compared to rats administered with PBS. In rats administered with ASC-TM, a further inhibitory effect on the increase in α-SMA and colI expression was observed compared to rats administered with ASC-null.

[0130] As described above, it has become clear that ASC-TM has not only embolic and thrombotic inhibitory effects (e.g., therapeutic or preventive effects on cerebral infarction and disseminated intravascular coagulation), but also fibrotic inhibitory effects (e.g., therapeutic or preventive effects on pulmonary fibrosis, idiopathic pulmonary fibrosis, and liver cirrhosis).

[0131] <5. Test Results D> Following the IRI (ischemic time 60 minutes) of the left kidney as described in section <1-5> above, PBS, ASC-null, or ASC-TM (5.0 x 10) were administered via the abdominal artery. ５ Cells (a rat drug) was injected. Seven days after injection, the left kidney was retrieved and immunohistochemical staining was performed to evaluate CD3 (T cell marker), CD68 (M1 and M2 macrophage marker), and CD163 (M2 macrophage marker). The expression of CD3, CD68, and CD163 serves as an indicator of immunosuppression, the presence of pro-inflammatory macrophages, and the presence of anti-inflammatory macrophages, respectively.

[0132] For staining CD3, CD68, and CD163, the following primary antibodies were used: rabbit polyclonal anti-CD3 antibody (abcam, ab5690), rabbit polyclonal anti-CD68 antibody (abcam, ab125212), and rabbit polyclonal anti-CD163 antibody (abcam, ab182422), respectively.

[0133] The test results are shown in Figures 401 and 402.

[0134] As is clear from Figures 401 and 402, the number of cells expressing CD3 and CD68 increased significantly in rats administered with PBS. On the other hand, in rats administered with ASC-null, an inhibitory effect on the increase in the number of cells expressing CD3 and CD68 was observed compared to rats administered with PBS. In rats administered with ASC-TM, a further inhibitory effect on the increase in the number of cells expressing CD3 and CD68 was observed compared to rats administered with ASC-null. On the other hand, in rats administered with ASC-TM, the number of cells expressing CD163 increased significantly compared to rats administered with ASC-null.

[0135] <6. Test Results E> In accordance with Section <1-4> above, BMS-345541 (IKKβ inhibitor) was added to the culture medium for ASCs at concentrations of 0 μM, 1.0 μM, 2.5 μM, or 5.0 μM, and the ASCs were cultured for 24 hours. After that, the ASCs were collected, and the expression levels of TM mRNA and TM protein in the ASCs were confirmed by RT-PCR and Western blotting, respectively.

[0136] The test results are shown in Figures 5, 501 and 502.

[0137] As is clear from Figure 5, 501, BMS-345541 (an IKKβ inhibitor) increased the mRNA expression level of TM. Also, as is clear from Figure 5, 502, BMS-345541 (an IKKβ inhibitor) increased the protein expression level of TM.

[0138] <7. Test Results F> <7-1. Test-1> In accordance with the above <1-7> column, ASC-BMS (7.5 × 10) was stimulated intravenously with the IKKβ inhibitor BMS-345541. ６ Rats were administered cells (a rat drug). Subsequently, rat mortality rates were evaluated by intravenous ASC injection.

[0139] The test results are shown in Figure 6, section 601. In the group administered ASC-NS, 2 out of 12 rats died within 24 hours, and intravascular pulmonary thrombi were confirmed in 24 out of 120 fields of view by H&E staining. On the other hand, in the group administered ASC-BMS, no rats died within 24 hours, and intravascular pulmonary thrombi were confirmed in 10 out of 120 fields of view by H&E staining. The number of intravascular pulmonary thrombi was lower in the group administered ASC-BMS compared to the group administered ASC-NS.

[0140] <7-2. Test-2> Following the IRI (ischemic time 60 minutes) of the left kidney as described in section <1-5> above, PBS, ASC-NS, or ASC-BMS (5.0 x 10) were administered via the abdominal artery. ５Cells (a rat drug) was injected. Twenty-one days after injection, the left kidney was retrieved, and the expression levels of α-SMA and collagen type I (colI) in the retrieved left kidney were checked by Western blotting, immunohistochemical staining, and Masson's trichrome staining.

[0141] For staining α-SMA and colI, mouse monoclonal α-SMA antibody (Sigma-Aldrich, A2547) and rabbit monoclonal anti-collagen I antibody (abcam, ab34710) were used as primary antibodies, respectively.

[0142] The test results are shown in Figure 6, 602, Figure 7, 701, and Figure 7, 702.

[0143] Figure 6, item 602, shows the results of the Western blotting test. As is clear from Figure 6, item 602, the expression level of α-SMA increased significantly in rats administered with PBS. On the other hand, in rats administered with ASC-NS, an inhibitory effect on the increase in α-SMA expression was observed compared to rats administered with PBS. In rats administered with ASC-BMS, a further inhibitory effect on the increase in α-SMA expression was observed compared to rats administered with ASC-NS.

[0144] Figure 7, item 701, shows the results of immunohistochemical staining. As is clear from Figure 7, item 701, the expression levels of α-SMA and COLI increased significantly in rats administered with PBS. On the other hand, in rats administered with ASC-NS, an inhibitory effect on the increase in α-SMA and COLI expression was observed compared to rats administered with PBS. In rats administered with ASC-BMS, a further inhibitory effect on the increase in α-SMA and COLI expression was observed compared to rats administered with ASC-NS.

[0145] Figure 7, item 702, shows the results of the Masson's trichrome staining test. As is clear from Figure 7, item 702, the expression levels of α-SMA and colI increased significantly in rats administered with PBS. On the other hand, in rats administered with ASC-NS, an inhibitory effect on the increase in α-SMA and colI expression was observed compared to rats administered with PBS. In rats administered with ASC-BMS, a further inhibitory effect on the increase in α-SMA and colI expression was observed compared to rats administered with ASC-NS.

[0146] As described above, it has become clear that ASC-BMS has not only embolic and thrombotic inhibitory effects (e.g., therapeutic or preventive effects on cerebral infarction and disseminated intravascular coagulation), but also fibrotic inhibitory effects (e.g., therapeutic or preventive effects on pulmonary fibrosis, idiopathic pulmonary fibrosis, and liver cirrhosis).

[0147] <8. Test Results G> <8-1. Test 1> Cell markers expressing ASC-null and ASC-TM were identified by flow cytometry. Flow cytometry was performed according to general procedures.

[0148] For data analysis, we used BD FACSVerse (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and Flowjo software (Flowjo, LCC; Ashland, Oregon, USA).

[0149] Antibodies for detecting each cell marker include anti-human CD29 IgG antibody (BioLegend, 303001, 1:20, San Diego, CA, USA), anti-human CD44 IgG antibody (BioLegend, 338804, 1:20), anti-human CD73 IgG antibody (BioLegend, 344004, 1:20), anti-human CD90 IgG antibody (BioLegend, 328108, 1:20), anti-human CD11b IgG antibody (BioLegend, 301404, 1:20), anti-human CD45 IgG antibody (BioLegend, 304006, 1:20), anti-human HLA-A, B, C IgG antibody (BioLegend, 311404, 1:20), and anti-human HLA-DR IgG. An antibody (BioLegend, 307604, 1:20) was used.

[0150] The test results are shown in Figure 8, item 801. As is clear from Figure 8, item 801, ASC-null and ASC-TM expressed standard MSC markers (e.g., CD29, CD44, CD73, CD90, HLA-A, B, C) and did not express MSC-negative markers (e.g., CD11b, CD34, CD45, HLA-DR). This indicates that the expression of MSC surface markers does not change due to AAV infection or other factors.

[0151] <8-2. Test 2> In accordance with Section <1-8> above, adipogenic differentiation induction and myelogenic differentiation induction were performed on ASC-null and ASC-TM, and the adipogenic and osteogenic capabilities of ASC-null and ASC-TM were evaluated.

[0152] The test results are shown in Figure 8, 802. As is clear from Figure 8, 802, both ASC-null and ASC-TM were found to possess adipogenesis and osteogenesis capabilities.

[0153] <9. Test Results H> <9-1. Test 1> Cell markers expressing ASC-NS and ASC-BMS were identified by flow cytometry. Flow cytometry was performed according to general procedures.

[0154] For data analysis, we used BD FACSVerse (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and Flowjo software (Flowjo, LCC; Ashland, Oregon, USA).

[0155] Antibodies for detecting each cell marker include anti-human CD29 IgG antibody (BioLegend, 303001, 1:20, San Diego, CA, USA), anti-human CD44 IgG antibody (BioLegend, 338804, 1:20), anti-human CD73 IgG antibody (BioLegend, 344004, 1:20), anti-human CD90 IgG antibody (BioLegend, 328108, 1:20), anti-human CD11b IgG antibody (BioLegend, 301404, 1:20), anti-human CD45 IgG antibody (BioLegend, 304006, 1:20), anti-human HLA-A, B, C IgG antibody (BioLegend, 311404, 1:20), and anti-human HLA-DR IgG. An antibody (BioLegend, 307604, 1:20) was used.

[0156] The test results are shown in Figure 9, 901. As is clear from Figure 9, 901, ASC-NS and ASC-BMS expressed standard MSC markers (e.g., CD29, CD44, CD73, CD90, HLA-A, B, C) and did not express MSC-negative markers (e.g., CD11b, CD34, CD45, HLA-DR). This indicates that stimulation with thrombomodulin expression inducers does not alter the expression of MSC surface markers.

[0157] <9-2. Test 2> In accordance with section <1-8> above, adipogenic differentiation induction and myelogenic differentiation induction were performed on ASC-NS and ASC-BMS, and the adipogenic and osteogenic capabilities of ASC-NS and ASC-BMS were evaluated.

[0158] The test results are shown in Figure 9, 902. As is clear from Figure 9, 902, both ASC-NS and ASC-BMS were found to possess adipogenesis and osteogenesis capabilities.

[0159] <10. Experiment I> RNA was extracted from ASCs according to conventional methods. Three biological copies were prepared in both ASC-BMS and ASC-NS. RNA sequencing was performed at Macrogen Japan (Tokyo, Japan).

[0160] The test results are shown in Figures 10-12. According to the Volcano plot, of the 19,025 quantitatively detected genes, 1,132 DEGs were identified between ASC-BMS and ASC-NS. Among these DEGs, 668 showed significantly increased expression and 464 showed significantly decreased expression.

[0161] A heatmap of DEGs created by hierarchical clustering revealed clear differences in mRNA expression between ASC-BMS and ASC-NS.

[0162] Similar to the RT-PCR results, ASC-BMS significantly increased TM mRNA expression, but did not affect the mRNA expression of complement and coagulation cascade-related genes, including coagulation factors such as TF, PAI-1, and protease-activated receptor (PAR1). In ASC-BMS, VEGFA mRNA expression was significantly increased, but did not affect the mRNA expression of TSG-6, IL-10, and HGF.

[0163] These data suggest that ASC-BMS carries a low risk of impairing the therapeutic effect of ASCs against tissue damage (e.g., multiple organ failure).

[0164] This invention can be used for the treatment and prevention of various diseases.

Claims

1. A pharmaceutical composition containing mesenchymal stem cells that overexpress thrombomodulin on the surface of the cells as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is used for the treatment or prevention of at least one selected from the group consisting of embolism, thrombosis, inflammation, fibrosis, organ damage, tissue damage, immunosuppression, and apoptosis enhancement.

3. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is used for the treatment or prevention of cerebral infarction, acute kidney injury, chronic kidney disease, sepsis, disseminated intravascular coagulation syndrome, pulmonary fibrosis, idiopathic pulmonary fibrosis, ARDS, cirrhosis, alcoholic hepatitis, non-alcoholic steatohepatitis, or multiple organ failure.

4. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered intravascularly.

5. The pharmaceutical composition according to claim 1, wherein the mesenchymal stem cells are cells in which thrombomodulin expression is not reduced regardless of the number of cell divisions or passages.

6. The pharmaceutical composition according to claim 1, wherein the mesenchymal stem cells are mesenchymal stem cells into which a thrombomodulin expression vector has been introduced, or mesenchymal stem cells after contact with a thrombomodulin expression inducer.

7. The pharmaceutical composition according to claim 6, wherein the expression inducer is BMS-345541, an IKKβ inhibitor, a compound having an imidazopyrazine skeleton in its molecule, an IκB inducer, or a compound having a flavonoid skeleton in its molecule.

8. The pharmaceutical composition according to claim 1, wherein the mesenchymal stem cells are mesenchymal stem cells cultured in a serum-free medium.

9. A method for producing a pharmaceutical composition, comprising a cell manufacturing step for producing mesenchymal stem cells that overexpress thrombomodulin on the surface of the cells, as an active ingredient of the pharmaceutical composition.

10. A method for producing the pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is used for the treatment or prevention of at least one selected from the group consisting of embolism, thrombosis, inflammation, fibrosis, organ damage, tissue damage, immunosuppression, and apoptosis enhancement.

11. A method for producing the pharmaceutical composition according to claim 10, wherein the pharmaceutical composition is used for the treatment or prevention of cerebral infarction, acute kidney injury, chronic kidney disease, sepsis, disseminated intravascular coagulation syndrome, pulmonary fibrosis, idiopathic pulmonary fibrosis, ARDS, cirrhosis of the liver, alcoholic hepatitis, non-alcoholic steatohepatitis, or multiple organ failure.

12. The method for producing the pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is administered intravascularly.

13. The method for producing the pharmaceutical composition according to claim 9, wherein the mesenchymal stem cells are cells in which thrombomodulin expression is not reduced regardless of the number of cell divisions or passages.

14. The method for producing a pharmaceutical composition according to claim 9, wherein the cell production step comprises introducing a vector for thrombomodulin expression into mesenchymal stem cells, or contacting mesenchymal stem cells with a thrombomodulin expression inducer.

15. The method for producing the pharmaceutical composition according to claim 14, wherein the expression inducer is BMS-345541, an IKKβ inhibitor, a compound having an imidazopyrazine skeleton in its molecule, an IκB inducer, or a compound having a flavonoid skeleton in its molecule.

16. The method for producing a pharmaceutical composition according to claim 9, wherein the cell production step comprises culturing mesenchymal stem cells in a serum-free medium.

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